Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein

ABSTRACT

The present invention relates to a nucleic add sequence, comprising or coding for a coding region, encoding at least one peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, at least one histone stem-loop and a poly(A) sequence or a polyadenylation signal. Furthermore the present invention provides the use of the nucleic add for increasing the expression of the encoded peptide or protein, particularly for the use in gene therapy. It also discloses its use for the preparation of a pharmaceutical composition, e.g., for use in gene therapy, particularly in the treatment of diseases which are in need of a treatment with a therapeutic peptide or protein, preferably as defined herein. The present invention further describes a method for increasing the expression of a peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, using the nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal.

This application is a continuation of U.S. application Ser. No.14/378,606, filed Aug. 13, 2014, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/EP2013/000461, filed Feb. 15, 2013, which is a continuation ofInternational Application No. PCT/EP2012/000671, filed Feb. 15, 2012.The entire text of each of the above referenced disclosures isspecifically incorporated herein by reference.

The present invention relates to a nucleic acid sequence, comprising orcoding for a coding region, encoding at least one peptide or proteincomprising a therapeutic protein or a fragment, variant or derivativethereof, at least one histone stem-loop and a poly(A) sequence or apolyadenylation signal. Furthermore, the present invention provides theuse of the nucleic acid for increasing the expression of said encodedpeptide or protein, particularly for the use in gene therapy. It alsodiscloses its use for the preparation of a pharmaceutical composition,e.g. for use in gene therapy, particularly in the treatment of diseaseswhich are in need of a treatment with a therapeutic peptide or protein,preferably as defined herein. The present invention further describes amethod for increasing the expression of a peptide or protein comprisinga therapeutic protein or a fragment, variant or derivative thereof,using the nucleic acid comprising or coding for a histone stem-loop anda poly(A) sequence or a polyadenylation signal.

Gene therapy means the use of nucleic acids as a pharmaceutical agent totreat a disease. It derives its name from the idea that nucleic acidscan be used to supplement or alter the expression of a gene within anindividual's cells as a therapy to treat a disease. The most common formof gene therapy involves using nucleic acids that encode a functional,therapeutic protein in order to replace a mutated gene. Other formsinvolve direct correction of a mutation, or using nucleic acids thatencode a therapeutic protein drug to provide treatment.

Gene therapy is a method of molecular medicine which already has beenproven in the therapy and prevention of diseases and generally exhibitsa considerable effect on daily medical practice, in particular on thetreatment of diseases as mentioned herein. Gene therapy is based on theintroduction of nucleic acids into the patient's cells or tissue andsubsequent processing of the information coded for by the nucleic acidthat has been introduced into the cells or tissue, that is to say the(protein) expression of the desired polypeptides.

Gene therapy may be beneficial for a lot of inherited or acquireddiseases, inter alia infectious diseases, neoplasms (e.g. cancer ortumour diseases), diseases of the blood and blood-forming organs,endocrine, nutritional and metabolic diseases, diseases of the nervoussystem, diseases of the circulatory system, diseases of the respiratorysystem, diseases of the digestive system, diseases of the skin andsubcutaneous tissue, diseases of the musculoskeletal system andconnective tissue, and diseases of the genitourinary system.

In gene therapy approaches, typically DNA is used even though RNA isalso known in recent developments. Importantly, in all these genetherapy approaches mRNA functions as messenger for the sequenceinformation of the encoded protein, irrespectively if DNA, viral RNA ormRNA is used.

In general RNA is considered an unstable molecule: RNases are ubiquitousand notoriously difficult to inactivate. Furthermore, RNA is alsochemically more labile than DNA. Thus, it is perhaps surprising that the“default state” of an mRNA in a eukaryotic cell is characterized by arelative stability and specific signals are required to accelerate thedecay of individual mRNAs. The main reason for this finding appears tobe that mRNA decay within cells is catalyzed almost exclusively byexonucleases. However, the ends of eukaryotic mRNAs are protectedagainst these enzymes by specific terminal structures and theirassociated proteins: a m7GpppN CAP at the 5′ end and typically a poly(A)sequence at the 3′ end. Removal of these two terminal modifications isthus considered rate limiting for mRNA decay. Although a stabilizingelement has been characterized in the 3′ UTR of the alpha-globin mRNA,RNA sequences affecting turnover of eukaryotic mRNAs typically act as apromoter of decay usually by accelerating deadenylation (reviewed inMeyer, S., C. Temme, et al. (2004), Crit Rev Biochem Mol Biol 39(4):197-216.).

As mentioned above, the 5′ ends of eukaryotic mRNAs are typicallymodified posttranscriptionally to carry a methylated CAP structure, e.g.m7GpppN. Aside from roles in RNA splicing, stabilization, and transport,the CAP structure significantly enhances the recruitment of the 40Sribosomal subunit to the 5′ end of the mRNA during translationinitiation. The latter function requires recognition of the CAPstructure by the eukaryotic initiation factor complex eIF4F. The poly(A)sequence additionally stimulates translation via increased 40S subunitrecruitment to mRNAs, an effect that requires the intervention ofpoly(A) binding protein (PABP). PABP, in turn, was recently demonstratedto interact physically with eIF4G, which is part of the CAP-bound eIF4Fcomplex. Thus, a closed loop model of translation initiation on capped,polyadenylated mRNAs was postulated (Michel, Y. M., D. Poncet, et al.(2000), J Biol Chem 275(41): 32268-76.).

Nearly all eukaryotic mRNAs end with such a poly(A) sequence that isadded to their 3′ end by the ubiquitous cleavage/polyadenylationmachinery. The presence of a poly(A) sequence at the 3′ end is one ofthe most recognizable features of eukaryotic mRNAs. After cleavage, mostpre-mRNAs, with the exception of replication-dependent histonetranscripts, acquire a polyadenylated tail. In this context, 3′ endprocessing is a nuclear co-transcriptional process that promotestransport of mRNAs from the nucleus to the cytoplasm and affects thestability and the translation of mRNAs. Formation of this 3′ end occursin a two step reaction directed by the cleavage/polyadenylationmachinery and depends on the presence of two sequence elements in mRNAprecursors (pre-mRNAs); a highly conserved hexanucleotide AAUAAA(polyadenylation signal) and a downstream G/U-rich sequence. In a firststep, pre-mRNAs are cleaved between these two elements. In a second steptightly coupled to the first step the newly formed 3′ end is extended byaddition of a poly(A) sequence consisting of 200-250 adenylates whichaffects subsequently all aspects of mRNA metabolism, including mRNAexport, stability and translation (Dominski, Z. and W. F. Marzluff(2007), Gene 396(2): 373-90.).

The only known exception to this rule are the replication-dependenthistone mRNAs which end with a histone stem-loop instead of a poly(A)sequence. Exemplary histone stem-loop sequences are described in Lopezet al. (Dávila López, M., & Samuelsson, T. (2008), RNA (New York, N.Y.),14(1), 1-10. doi:10.1261/rna.782308.).

The stem-loops in histone pre-mRNAs are typically followed by apurine-rich sequence known as the histone downstream element (HDE).These pre-mRNAs are processed in the nucleus by a single endonucleolyticcleavage approximately 5 nucleotides downstream of the stem-loop,catalyzed by the U7 snRNP through base pairing of the U7 snRNA with theHDE. The 3′-UTR sequence comprising the histone stem-loop structure andthe histone downstream element (HDE) (binding site of the U7 snRNP) wereusually termed as histone 3′-processing signal (see e.g. Chodchoy, N.,N. B. Pandey, et al. (1991). Mol Cell Biol 11(1): 497-509.).

Due to the requirement to package newly synthesized DNA into chromatin,histone synthesis is regulated in concert with the cell cycle. Increasedsynthesis of histone proteins during S phase is achieved bytranscriptional activation of histone genes as well asposttranscriptional regulation of histone mRNA levels. It could be shownthat the histone stem-loop is essential for all posttranscriptionalsteps of histone expression regulation. It is necessary for efficientprocessing, export of the mRNA into the cytoplasm, loading ontopolyribosomes, and regulation of mRNA stability.

In the above context, a 32 kDa protein was identified, which isassociated with the histone stem-loop at the 3′-end of the histonemessages in both the nucleus and the cytoplasm. The expression level ofthis stem-loop binding protein (SLBP) is cell-cycle regulated and ishighest during S-phase when histone mRNA levels are increased. SLBP isnecessary for efficient 3′-end processing of histone pre-mRNA by the U7snRNP. After completion of processing, SLBP remains associated with thestem-loop at the end of mature histone mRNAs and stimulates theirtranslation into histone proteins in the cytoplasm. (Dominski, Z. and W.F. Marzluff (2007), Gene 396(2): 373-90). Interestingly, the RNA bindingdomain of SLBP is conserved throughout metazoa and protozoa (DavilaLopez, M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308) and it could be shown that its binding to thehistone stem-loop sequence is dependent on the stem-loop structure andthat the minimum binding site contains at least 3 nucleotides 5′ and 2nucleotides 3′ of the stem-loop (Pandey, N. B., et al. (1994), Molecularand Cellular Biology, 14(3), 1709-1720 and Williams, A. S., & Marzluff,W. F., (1995), Nucleic Acids Research, 23(4), 654-662.).

Even though histone genes are generally classified as either“replication-dependent”, giving rise to mRNA ending in a histonestem-loop, or “replacement-type”, giving rise to mRNA bearing apoly(A)-tail instead, naturally occurring mRNAs containing both ahistone stem-loop and poly(A) or oligo(A) 3′ thereof have beenidentified in some very rare cases. Sanchez et al. examined the effectof naturally occurring oligo(A) tails appended 3′ of the histonestem-loop of histone mRNA during Xenopus oogenesis using Luciferase as areporter protein and found that the oligo(A) tail is an active part ofthe translation repression mechanism that silences histone mRNA duringoogenesis and its removal is part of the mechanism that activatestranslation of histone mRNAs (Sanchez, R. and W. F. Marzluff (2004), MolCell Biol 24(6): 2513-25).

Furthermore, the requirements for regulation of replication dependenthistones at the level of pre-mRNA processing and mRNA stability havebeen investigated using artificial constructs coding for the markerprotein alpha Globin, taking advantage of the fact that the globin genecontains introns as opposed to the intron-less histone genes. For thispurpose constructs were generated in which the alpha globin codingsequence was followed by a histone stem-loop signal (histone stem-loopfollowed by the histone downstream element) and a polyadenylation signal(Whitelaw, E., et al. (1986). Nucleic Acids Research, 14(17),7059-7070.; Pandey, N. B., & Marzluff, W. F. (1987). Molecular andCellular Biology, 7(12), 4557-4559.; Pandey, N. B., et al. (1990).Nucleic Acids Research, 18(11), 3161-3170).

In another approach Lüscher et al. investigated the cell-cycle dependentregulation of a recombinant histone H4 gene. Constructs were generatedin which the H4 coding sequence was followed by a histone stem-loopsignal and a polyadenylation signal, the two processing signalsincidentally separated by a galactokinase coding sequence (Liischer, B.et al., (1985). Proc. Natl. Acad. Sci. USA, 82(13), 4389-4393).

Additionally, Stauber et al. identified the minimal sequence required toconfer cell-cycle regulation on histone H4 mRNA levels. For theseinvestigations constructs were used, comprising a coding sequence forthe selection marker Xanthine:guanine phosphoribosyl transferase (GPT)preceding a histone stem-loop signal followed by a polyadenylationsignal (Stauber, C. et al., (1986). EMBO J, 5(12), 3297-3303).

Examining histone pre-mRNA processing Wagner et al. identified factorsrequired for cleavage of histone pre-mRNAs using a reporter constructplacing EGFP between a histone stem-loop signal and a polyadenylationsignal, such that EGFP was expressed only in case histone pre-mRNAprocessing was disrupted (Wagner, E. J. et al., (2007). Mol Cell 28(4),692-9).

To be noted, translation of polyadenylated mRNA usually requires the 3′poly(A) sequence to be brought into proximity of the 5′ CAP. This ismediated through protein-protein interaction between the poly(A) bindingprotein and eukaryotic initiation factor eIF4G. With respect toreplication-dependent histone mRNAs, an analogous mechanism has beenuncovered. In this context, Gallie et al. show that the histonestem-loop is functionally similar to a poly(A) sequence in that itenhances translational efficiency and is co-dependent on a 5′-CAP inorder to establish an efficient level of translation. They showed thatthe histone stem-loop is sufficient and necessary to increase thetranslation of a reporter mRNA in transfected Chinese hamster ovarycells but must be positioned at the 3′-terminus in order to functionoptimally. Therefore, similar to the poly(A) tail on other mRNAs, the 3′end of these histone mRNAs appears to be essential for translation invivo and is functionally analogous to a poly(A) tail (Gallie, D. R.,Lewis, N. J., & Marzluff, W. F. (1996), Nucleic Acids Research, 24(10),1954-1962).

Additionally, it could be shown that SLBP is bound to the cytoplasmichistone mRNA and is required for its translation. Even though SLBP doesnot interact directly with eIF4G, the domain required for translation ofhistone mRNA interacts with the recently identified protein SLIP1. In afurther step, SLIP1 interacts with eIF4G and allows to circularizehistone mRNA and to support efficient translation of histone mRNA by amechanism similar to the translation of polyadenylated mRNAs.

As mentioned above, gene therapy approaches normally use DNA to transferthe coding information into the cell which is then transcribed intomRNA, carrying the naturally occurring elements of an mRNA, particularlythe 5′-CAP structure and the 3′ poly(A) sequence to ensure expression ofthe encoded therapeutic or antigenic protein.

However, in many cases expression systems based on the introduction ofsuch nucleic acids into the patient's cells or tissue and the subsequentexpression of the desired polypeptides coded for by these nucleic acidsdo not exhibit the desired, or even the required, level of expressionwhich may allow for an efficient therapy, irrespective as to whether DNAor RNA is used.

In the prior art, different attempts have hitherto been made to increasethe yield of the expression of an encoded protein, in particular by useof improved expression systems, both in vitro and/or in vivo. Methodsfor increasing expression described generally in the prior art areconventionally based on the use of expression vectors or cassettescontaining specific promoters and corresponding regulation elements. Asthese expression vectors or cassettes are typically limited toparticular cell systems, these expression systems have to be adapted foruse in different cell systems. Such adapted expression vectors orcassettes are then usually transfected into the cells and typicallytreated in dependence of the specific cell line. Therefore, preferenceis given primarily to those nucleic acid molecules which are able toexpress the encoded proteins in a target cell by systems inherent in thecell, independent of promoters and regulation elements which arespecific for particular cell types. In this context, there can bedistinguished between mRNA stabilizing elements and elements whichincrease translation efficiency of the mRNA.

mRNAs which are optimized in their coding sequence and which are ingeneral suitable for such a purpose are described in application WO02/098443 (CureVac GmbH). For example, WO 02/098443 describes mRNAs thatare stabilised in general form and optimised for translation in theircoding regions. WO 02/098443 further discloses a method for determiningsequence modifications. WO 02/098443 additionally describespossibilities for substituting adenine and uracil nucleotides in mRNAsequences in order to increase the guanine/cytosine (G/C) content of thesequences. According to WO 02/098443, such substitutions and adaptationsfor increasing the G/C content can be used for gene therapeuticapplications but also genetic vaccines in the treatment of cancer orinfectious diseases. In this context, WO 02/098443 generally mentionssequences as a base sequence for such modifications, in which themodified mRNA codes for at least one biologically active peptide orpolypeptide, which is translated in the patient to be treated, forexample, either not at all or inadequately or with faults.Alternatively, WO 02/098443 proposes mRNAs coding for antigens e.g.therapeutic proteins or viral antigens as a base sequence for suchmodifications.

In a further approach to increase the expression of an encoded proteinthe application WO 2007/036366 describes the positive effect of longpoly(A) sequences (particularly longer than 120 bp) and the combinationof at least two 3′ untranslated regions of the beta globin gene on mRNAstability and translational activity.

However, even though all these latter prior art documents already try toprovide quite efficient tools for gene therapy approaches andadditionally improved mRNA stability and translational activity, therestill remains the problem of a generally lower stability of RNA-basedapplications versus DNA vaccines and DNA based gene therapeuticapproaches. Accordingly, there still exists a need in the art to provideimproved tools for gene therapy approaches or as a supplementary therapyfor conventional treatments as discussed above, which allow for betterprovision of encoded proteins in vivo, e.g. via a further improved mRNAstability and/or translational activity, preferably for gene therapy.

Furthermore despite of all progress in the art, efficient expression ofan encoded peptide or protein in cell-free systems, cells or organisms(recombinant expression) is still a challenging problem.

The object underlying the present invention is, therefore, to provideadditional and/or alternative methods to increase expression of anencoded protein, preferably via further stabilization of the mRNA and/oran increase of the translational efficiency of such an mRNA with respectto such nucleic acids known from the prior art for the use in genetherapy in the therapeutic or prophylactic treatment of inherited oracquired diseases, particulary as defined herein.

This object is solved by the subject matter of the attached claims.Particularly, the object underlying the present invention is solvedaccording to a first aspect by an inventive nucleic acid sequencecomprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein        which comprises a therapeutic protein or a fragment, variant or        derivative thereof;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal,

preferably for increasing the expression of said encoded peptide orprotein.

Alternatively, any appropriate stem loop sequence other than a histonestem loop sequence (derived from histone genes, in particular histonegenes of the families H1, H2A, H2B, H3 and H4) may be employed by thepresent invention in all of its aspects and embodiments.

In this context it is particularly preferred that the inventive nucleicacid according to the first aspect of the present invention is producedat least partially by DNA or RNA synthesis, preferably as describedherein or is an isolated nucleic acid.

The present invention is based on the surprising finding of the presentinventors, that the combination of a poly(A) sequence or polyadenylationsignal and at least one histone stem-loop, even though both representingalternative mechanisms in nature, acts synergistically as thiscombination increases the protein expression manifold above the levelobserved with either of the individual elements. The synergistic effectof the combination of poly(A) and at least one histone stem-loop is seenirrespective of the order of poly(A) and histone stem-loop andirrespective of the length of the poly(A) sequence.

Therefore it is particularly preferred that the inventive nucleic acidsequence comprises or codes for a) a coding region, encoding at leastone peptide or protein which comprises a therapeutic protein or afragment, variant or derivative thereof; b) at least one histonestem-loop, and c) a poly(A) sequence or polyadenylation sequence;preferably for increasing the expression level of said encoded peptideor protein, wherein the encoded protein is preferably no histoneprotein, in particular no histone protein of the H4, H3, H2A and/or H2Bhistone family or a fragment, derivative or variant thereof retaininghistone(-like) function), namely forming a nucleosome. Also, the encodedprotein typically does not correspond to a histone linker protein of theH1 histone family. The inventive nucleic acid molecule does typicallynot contain any regulatory signals (5′ and/or, particularly, 3′) of amouse histone gene, in particular not of a mouse histone gene H2A and,further, most preferably not of the mouse histone gene H2A614. Inparticular, it does not contain a histone stem loop and/or a histonestem loop processing signal from a mouse histone gene, in particular notof mouse histone gene H2A und, most preferably not of mouse histone geneH2A614.

Also, the inventive nucleic acid typically does not provide a reporterprotein (e.g. Luciferase, GFP, EGFP, β-Galactosidase, particularlyEGFP), galactokinase (galK) and/or marker or selection protein (e.g.alpha-Globin, Galactokinase and Xanthine:Guanine phosphoribosyltransferase (GPT)) or a bacterial reporter protein, e.g. chloramphenicolacetyl transferase (CAT) or other bacterial antibiotics resistanceproteins, e.g. derived from the bacterial neo gene in its element (a).

A reporter, marker or selection protein is typically understood not tobe a therapeutic protein according to the invention. A reporter, markeror selection protein or its underlying gene is commonly used as aresearch tool in bacteria, cell culture, animals or plants. They conferon organisms (preferably heterologously) expressing them an easilyidentifiable property, which may be measured or which allows forselection. Specifically, marker or selection proteins exhibit aselectable function. Typically, such selection, marker or reporterproteins do not naturally occur in humans or other mammals, but arederived from other organisms, in particular from bacteria or plants.Accordingly, proteins with selection, marker or reporter functionoriginating from species other than mammals, in particular other thanhumans, are preferably excluded from being understood as “therapeuticprotein” according to the present invention. In particular, a selection,marker or reporter protein allows to identify transformed cells by invitro assays based e.g. on fluorescence or other spectroscopictechniques and resistance towards antibiotics. Selection, reporter ormarker genes awarding such properties to transformed cells are thereforetypically not understood to be a therapeutic protein according to theinvention.

In any case, reporter, marker or selection proteins do usually not exertany therapeutic effect. If any single reporter, marker or selectionprotein should nevertheless do so (in addition to its reporter,selection or marker function), such a reporter, marker or selectionprotein is preferably not understood to be a “therapeutic protein”within the meaning of the present invention.

In contrast, a therapeutic protein (including its fragments, variantsand derivatives), in particular excluding histone genes of the familiesH1, H2A, H2B, H3 and H4, according to the present invention doestypically not exhibit a selection, marker or reporter function. If anysingle “therapeutic protein” nevertheless should do so (in addition toits therapeutic function), such a therapeutic protein is preferably notunderstood to be a “selection, marker or reporter protein” within themeaning of the present invention.

It is most preferably understood that a therapeutic protein according tothe invention is derived from mammals, in particular humans, and doesnot qualify as selection, marker or reporter protein.

Accordingly, it is preferred that the coding region (a) encoding atleast one peptide or protein is heterologous to at least (b) the atleast one histone stem loop, or more broadly, to any appropriate stemloop. In other words, “heterologous” in the context of the presentinvention means that the at least one stem loop sequence does notnaturally occur as a (regulatory) sequence (e.g. at the 3′UTR) of thespecific gene, which encodes the (therapeutic) protein or peptide ofelement (a) of the inventive nucleic acid. Accordingly, the (histone)stem loop of the inventive nucleic acid is derived preferably from the3′ UTR of a gene other than the one comprising the coding region ofelement (a) of the inventive nucleic acid. E.g., the coding region ofelement (a) will not encode a histone protein or a fragment, variant orderivative thereof (retaining the function of a histone protein), if theinventive nucleic is heterologous, but will encode any other peptide orsequence (of the same or another species) which exerts a biologicalfunction, preferably therapeutic function other than a histone(-like)function, e.g. will encode an therapeutic protein (exerting atherapeutic function, e.g. in terms endocrine disorders, replacingdefective endogenous, e.g. mammalian, in particular human proteins).

In this context it is particularly preferred that the inventive nucleicacid comprises or codes for in 5′- to 3′-direction:

-   -   a) a coding region, encoding at least one peptide or protein        which comprises a therapeutic protein or a fragment, variant or        derivative thereof;    -   b) at least one histone stem-loop, optionally without a histone        downstream element (HDE) 3′ to the histone stem-loop    -   c) a poly(A) sequence or a polyadenylation signal.

The term “histone downstream element (HDE) refers to a purine-richpolynucleotide stretch of about 15 to 20 nucleotides 3′ of naturallyoccurring histone stem-loops, which represents the binding site for theU7 snRNA involved in processing of histone pre-mRNA into mature histonemRNA. For example in sea urchins the HDE is CAAGAAAGA (Dominski, Z. andW. F. Marzluff (2007), Gene 396(2): 373-90).

Furthermore it is preferable that the inventive nucleic acid accordingto the first aspect of the present invention does not comprise anintron.

In another particular preferred embodiment, the inventive nucleic acidsequence according to the first aspect of the present inventioncomprises or codes for from 5′ to 3′:

-   -   a) a coding region, preferably encoding at least one peptide or        protein which comprises a therapeutic protein or a fragment,        variant or derivative thereof;    -   c) a poly(A) sequence; and    -   b) at least one histone stem-loop.

The inventive nucleic acid sequence according to the first embodiment ofthe present invention comprise any suitable nucleic acid, selected e.g.from any (single-stranded or double-stranded) DNA, preferably, withoutbeing limited thereto, e.g. genomic DNA, plasmid DNA, single-strandedDNA molecules, double-stranded DNA molecules, or may be selected e.g.from any PNA (peptide nucleic acid) or may be selected e.g. from any(single-stranded or double-stranded) RNA, preferably a messenger RNA(mRNA); etc. The inventive nucleic acid sequence may also comprise aviral RNA (vRNA). However, the inventive nucleic acid sequence may notbe a viral RNA or may not contain a viral RNA. More specifically, theinventive nucleic acid sequence may not contain viral sequence elements,e.g. viral enhancers or viral promotors (e.g. no inactivated viralpromoter or sequence elements, more specifically not inactivated byreplacement strategies), or other viral sequence elements, or viral orretroviral nucleic acid sequences. More specifically, the inventivenucleic acid sequence may not be a retroviral or viral vector or amodified retroviral or viral vector.

In any case, the inventive nucleic acid sequence may or may not containan enhancer and/or promoter sequence, which may be modified or not orwhich may be activated or not. The enhancer and or promoter may be plantexpressible or not expressible, and/or in eukaryotes expressible or notexpressible and/or in prokaryotes expressible or not expressible. Theinventive nucleic acid sequence may contain a sequence encoding a(self-splicing) ribozyme or not.

In specific embodiments the inventive nucleic acid sequence may be ormay comprise a self-replicating RNA (replicon).

Preferably, the inventive nucleic acid sequence is a plasmid DNA, or anRNA, particularly an mRNA.

In particular embodiments of the first aspect of the present invention,the inventive nucleic acid is a nucleic acid sequence comprised in anucleic acid suitable for in vitro transcription, particularly in anappropriate in vitro transcription vector (e.g. a plasmid or a linearnucleic acid sequence comprising specific promoters for in vitrotranscription such as T3, T7 or Sp6 promoters).

In further particular preferred embodiments of the first aspect of thepresent invention, the inventive nucleic acid is comprised in a nucleicacid suitable for transcription and/or translation in an expressionsystem (e.g. in an expression vector or plasmid), particularly aprokaryotic (e.g. bacteria like E. coli) or eukaryotic (e.g. mammaliancells like CHO cells, yeast cells or insect cells or whole organismslike plants or animals) expression system.

The term “expression system” means a system (cell culture or wholeorganisms) which is suitable for production of peptides, proteins or RNAparticularly mRNA (recombinant expression).

The inventive nucleic acid sequence according to the first aspect of thepresent invention comprises or codes for at least one (histone)stem-loop. A stem-loop, in general (irrespective of whether it is ahistone stem loop or not), can occur in single-stranded DNA or, morecommonly, in RNA. The structure is also known as a hairpin or hairpinloop and usually consists of a stem and a (terminal) loop within aconsecutive sequence, wherein the stem is formed by two neighboredentirely or partially reverse complementary sequences separated by ashort sequence as sort of spacer, which builds the loop of the stem-loopstructure. The two neighbored entirely or partially reversecomplementary sequences may be defined as e.g. stem loop elements stem1and stem2. The stem loop is formed when these two neighbored entirely orpartially reverse complementary sequences, e.g. stem loop elements stem1and stem2, form base-pairs with each other, leading to a double strandednucleic acid sequence stretch comprising an unpaired loop at itsterminal ending formed by the short sequence located between stem loopelements stem1 and stem2 on the consecutive sequence. The unpaired loopthereby typically represents a region of the nucleic acid which is notcapable of base pairing with either of these stem loop elements. Theresulting lollipop-shaped structure is a key building block of many RNAsecondary structures.

The formation of a stem-loop structure is thus dependent on thestability of the resulting stem and loop regions, wherein the firstprerequisite is typically the presence of a sequence that can fold backon itself to form a paired double strand. The stability of paired stemloop elements is determined by the length, the number of mismatches orbulges it contains (a small number of mismatches is typically tolerable,especially for a longer double stranded stretch), and the basecomposition of the paired region. In the context of the presentinvention, a loop length of 3 to 15 bases is conceivable, while a morepreferred loop length is 3-10 bases, more preferably 3 to 8, 3 to 7, 3to 6 or even more preferably 4 to 5 bases, and most preferably 4 bases.The stem sequence forming the double stranded structure typically has alength of between 5 to 10 bases, more preferably, between 5 to 8 bases.

In the context of the present invention, a histone stem-loop istypically derived from histone genes (e.g. genes from the histonefamilies H1, H2A, H2B, H3, H4) and comprises an intramolecular basepairing of two neighbored entirely or partially reverse complementarysequences, thereby forming a stem-loop. Typically, a histone 3′ UTRstem-loop is an RNA element involved in nucleocytoplasmic transport ofthe histone mRNAs, and in the regulation of stability and of translationefficiency in the cytoplasm. The mRNAs of metazoan histone genes lackpolyadenylation and a poly-A tail, instead 3′ end processing occurs at asite between this highly conserved stem-loop and a purine rich regionaround 20 nucleotides downstream (the histone downstream element, orHDE). The histone stem-loop is bound by a 31 kDa stem-loop bindingprotein (SLBP—also termed the histone hairpin binding protein, or HBP).Such histone stem-loop structures are preferably employed by the presentinvention in combination with other sequence elements and structures,which do not occur naturally (which means in untransformed livingorganisms/cells) in histone genes, but are combined—according to theinvention—to provide an artificial, heterologous nucleic acid.Accordingly, the present invention is particularly based on the findingthat an artificial (non-native) combination of a histone stem-loopstructure with other heterologous sequence elements, which do not occurin histone genes or metazoan histone genes and are isolated fromoperational and/or regulatory sequence regions (influencingtranscription and/or translation) of genes coding for proteins otherthan histones, provide advantageous effects. Accordingly, one aspect ofthe invention provides the combination of a histone stem-loop structurewith a poly(A) sequence or a sequence representing a polyadenylationsignal (3′-terminal of a coding region), which does not occur inmetazoan histone genes.

According to another preferred aspect of the invention, a combination ofa histone stem-loop structure with a coding region coding for atherapeutic protein, which does, preferably not occur in metazoanhistone genes, is provided herewith (coding region and histone stem loopsequence are heterologous). It is preferred, if such therapeuticproteins occur naturally in mammalians, preferably humans. In a stillfurther preferred embodiment, all the elements (a), (b) and (c) of theinventive nucleic acid are heterologous to each other and are combinedartificially from three different sources, e.g. (a) the therapeuticprotein coding region from a human gene, (b) the histone stem loop froman untranlated region of a metazoan, e.g. mammalian, non-human or human,histone gene and (c) the poly(A) sequence or the polyadenylation signalfrom e.g. an untranlated region of a gene other than a histone gene andother than the gene coding for the therapeutic protein according toelement (a) of such an inventive nucleic acid.

A histone stem loop is, therefore, a stem-loop structure as describedherein, which, if preferably functionally defined, exhibits/retains theproperty of binding to its natural binding partner, the stem-loopbinding protein (SLBP—also termed the histone hairpin binding protein,or HBP).

According to the present invention the histone stem loop sequenceaccording to component (b) of claim 1 may not derived from a mousehistone protein. More specifically, the histone stem loop sequence maynot be derived from mouse histone gene H2A614. Also, the nucleic acid ofthe invention may neither contain a mouse histone stem loop sequence norcontain mouse histone gene H2A614. Further, the inventive nucleic acidsequence may not contain a stem-loop processing signal, morespecifically, a mouse histone processing signal and, most specifically,may not contain mouse stem loop processing signal H2kA614, even if theinventive nucleic acid sequence may contain at least one mammalianhistone gene. However, the at least one mammalian histone gene may notbe Seq. ID No. 7 of WO 01/12824.

According to one preferred embodiment of the first inventive aspect, theinventive nucleic acid sequence comprises or codes for at least onehistone stem-loop sequence, preferably according to at least one of thefollowing formulae (I) or (II):

formula (I) (stem-loop sequence without stem bordering elements):

formula (II) (stem-loop sequence with stem bordering elements):

wherein:

-   stem1 or stem2 bordering elements N1-6 is a consecutive sequence of    1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more    preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each    N is independently from another selected from a nucleotide selected    from A, U, T, G and C, or a nucleotide analogue thereof;-   stem1 [N₀₋₂GN₃₋₅] is reverse complementary or partially reverse    complementary with element stem2, and is a consecutive sequence    between of 5 to 7 nucleotides;    -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of        0 to 1, more preferably of 1 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof;    -   wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of        4 to 5, more preferably of 4 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof, and    -   wherein G is guanosine or an analogue thereof, and may be        optionally replaced by a cytidine or an analogue thereof,        provided that its complementary nucleotide cytidine in stem2 is        replaced by guanosine;-   loop sequence [N₀₋₄(U/T)N₀₋₄] is located between elements stem1 and    stem2, and is a consecutive sequence of 3 to 5 nucleotides, more    preferably of 4 nucleotides;    -   wherein each N₀₋₄ is independent from another a consecutive        sequence of 0 to 4, preferably of 1 to 3, more preferably of 1        to 2 N, wherein each N is independently from another selected        from a nucleotide selected from A, U, T, G and C or a nucleotide        analogue thereof; and    -   wherein U/T represents uridine, or optionally thymidine;-   stem2 [N₃₋₅CN₀₋₂] is reverse complementary or partially reverse    complementary with element stem1, and is a consecutive sequence    between of 5 to 7 nucleotides;    -   wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of        4 to 5, more preferably of 4 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof;    -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of        0 to 1, more preferably of 1 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        or C or a nucleotide analogue thereof; and    -   wherein C is cytidine or an analogue thereof, and may be        optionally replaced by a guanosine or an analogue thereof        provided that its complementary nucleotide guanosine in stem1 is        replaced by cytidine;        wherein

stem1 and stem2 are capable of base pairing with each other forming areverse complementary sequence, wherein base pairing may occur betweenstem1 and stem2, e.g. by Watson-Crick base pairing of nucleotides A andU/T or G and C or by non-Watson-Crick base pairing e.g. wobble basepairing, reverse Watson-Crick base pairing, Hoogsteen base pairing,reverse Hoogsteen base pairing or are capable of base pairing with eachother forming a partially reverse complementary sequence, wherein anincomplete base pairing may occur between stem1 and stem2, on the basisthat one or more bases in one stem do not have a complementary base inthe reverse complementary sequence of the other stem.

In the above context, a wobble base pairing is typically anon-Watson-Crick base pairing between two nucleotides. The four mainwobble base pairs in the present context, which may be used, areguanosine-uridine, inosine-uridine, inosine-adenosine, inosine-cytidine(G-U/T, I-U/T, I-A and I-C) and adenosine-cytidine (A-C).

Accordingly, in the context of the present invention, a wobble base is abase, which forms a wobble base pair with a further base as describedabove. Therefore non-Watson-Crick base pairing, e.g. wobble basepairing, may occur in the stem of the histone stem-loop structureaccording to the present invention.

In the above context a partially reverse complementary sequencecomprises maximally 2, preferably only one mismatch in thestem-structure of the stem-loop sequence formed by base pairing of stem1and stem2. In other words, stem1 and stem2 are preferably capable of(full) base pairing with each other throughout the entire sequence ofstem1 and stem2 (100% of possible correct Watson-Crick ornon-Watson-Crick base pairings), thereby forming a reverse complementarysequence, wherein each base has its correct Watson-Crick ornon-Watson-Crick base pendant as a complementary binding partner.Alternatively, stem1 and stem2 are preferably capable of partial basepairing with each other throughout the entire sequence of stem1 andstem2, wherein at least about 70%, 75%, 80%, 85%, 90%, or 95% of the100% possible correct Watson-Crick or non-Watson-Crick base pairings areoccupied with the correct Watson-Crick or non-Watson-Crick base pairingsand at most about 30%, 25%, 20%, 15%, 10%, or 5% of the remaining basesare unpaired.

According to a preferred embodiment of the first inventive aspect, theat least one histone stem-loop sequence (with stem bordering elements)of the inventive nucleic acid sequence as defined herein comprises alength of about 15 to about 45 nucleotides, preferably a length of about15 to about 40 nucleotides, preferably a length of about 15 to about 35nucleotides, preferably a length of about 15 to about 30 nucleotides andeven more preferably a length of about 20 to about 30 and mostpreferably a length of about 24 to about 28 nucleotides.

According to a further preferred embodiment of the first inventiveaspect, the at least one histone stem-loop sequence (without stembordering elements) of the inventive nucleic acid sequence as definedherein comprises a length of about 10 to about 30 nucleotides,preferably a length of about 10 to about 20 nucleotides, preferably alength of about 12 to about 20 nucleotides, preferably a length of about14 to about 20 nucleotides and even more preferably a length of about 16to about 17 and most preferably a length of about 16 nucleotides.

According to a further preferred embodiment of the first inventiveaspect, the inventive nucleic acid sequence according to the firstaspect of the present invention may comprise or code for at least onehistone stem-loop sequence according to at least one of the followingspecific formulae (Ia) or (IIa):

formula (Ia) (stem-loop sequence without stem bordering elements):

formula (IIa) (stem-loop sequence with stem bordering elements):

wherein:

N, C, G, T and U are as defined above.

According to a further more particularly preferred embodiment of thefirst aspect, the inventive nucleic acid sequence may comprise or codefor at least one histone stem-loop sequence according to at least one ofthe following specific formulae (Ib) or (IIb):

formula (Ib) (stem-loop sequence without stem bordering elements):

formula (IIb) (stem-loop sequence with stem bordering elements):

wherein:

N, C, G, T and U are as defined above.

According to an even more preferred embodiment of the first inventiveaspect, the inventive nucleic acid sequence according to the firstaspect of the present invention may comprise or code for at least onehistone stem-loop sequence according to at least one of the followingspecific formulae (Ic) to (Ih) or (IIc) to (IIh), shown alternatively inits stem-loop structure and as a linear sequence representing histonestem-loop sequences as generated according to Example 1:

formula (Ic): (metazoan and protozoan histone stem-loop consensussequence without stem bordering elements):

formula (IIc): (metazoan and protozoan histone stem-loop consensussequence with stem bordering elements):

formula (Id): (without stem bordering elements)

formula (IId): (with stem bordering elements)

formula (Ie): (protozoan histone stem-loop consensus sequence withoutstem bordering elements)

formula (IIe): (protozoan histone stem-loop consensus sequence with stembordering elements)

formula (If): (metazoan histone stem-loop consensus sequence withoutstem bordering elements)

formula (IIf): (metazoan histone stem-loop consensus sequence with stembordering elements)

formula (Ig): (vertebrate histone stem-loop consensus sequence withoutstem bordering elements)

formula (IIg): (vertebrate histone stem-loop consensus sequence withstem bordering elements)

formula (Ih): (human histone stem-loop consensus sequence (Homo sapiens)without stem bordering elements)

formula (IIh): (human histone stem-loop consensus sequence (Homosapiens) with stem bordering elements)

wherein in each of above formulae (Ic) to (Ih) or (IIc) to (IIh):

-   N, C, G, A, T and U are as defined above;-   each U may be replaced by T;-   each (highly) conserved G or C in the stem elements 1 and 2 may be    replaced by its complementary nucleotide base C or G, provided that    its complementary nucleotide in the corresponding stem is replaced    by its complementary nucleotide in parallel; and/or

G, A, T, U, C, R, Y, M, K, S, W, H, B, V, D, and N are nucleotide basesas defined in the following Table:

abbreviation Nucleotide bases remark G G Guanine A A Adenine T T ThymineU U Uracile C C Cytosine R G or A Purine Y T/U or C Pyrimidine M A or CAmino K G or T/U Keto S G or C Strong (3H bonds) W A or T/U Weak (2Hbonds) H A or C or T/U Not G B G or T/U or C Not A V G or C or A Not T/UD G or A or T/U Not C N G or C or T/U or A Any base * Present or notBase may be present or not

In this context it is particularly preferred that the histone stem-loopsequence according to at least one of the formulae (I) or (Ia) to (Ih)or (II) or (IIa) to (IIh) of the present invention is selected from anaturally occurring histone stem loop sequence, more particularlypreferred from protozoan or metazoan histone stem-loop sequences, andeven more particularly preferred from vertebrate and mostly preferredfrom mammalian histone stem-loop sequences especially from human histonestem-loop sequences.

According to a particularly preferred embodiment of the first aspect,the histone stem-loop sequence according to at least one of the specificformulae (I) or (Ia) to (Ih) or (II) or (IIa) to (IIh) of the presentinvention is a histone stem-loop sequence comprising at each nucleotideposition the most frequently occurring nucleotide, or either the mostfrequently or the second-most frequently occurring nucleotide ofnaturally occurring histone stem-loop sequences in metazoa and protozoa(FIG. 1), protozoa (FIG. 2), metazoa (FIG. 3), vertebrates (FIG. 4) andhumans (FIG. 5) as shown in FIG. 1-5. In this context it is particularlypreferred that at least 80%, preferably at least 85%, or most preferablyat least 90% of all nucleotides correspond to the most frequentlyoccurring nucleotide of naturally occurring histone stem-loop sequences.

In a further particular embodiment of the first aspect, the histonestem-loop sequence according to at least one of the specific formulae(I) or (Ia) to (Ih) of the present invention is selected from followinghistone stem-loop sequences (without stem-bordering elements)representing histone stem-loop sequences as generated according toExample 1:

(SEQ ID NO: 13 according to formula (Ic)) VGYYYYHHTHRVVRCB(SEQ ID NO: 14 according to formula (Ic)) SGYYYTTYTMARRRCS(SEQ ID NO: 15 according to formula (Ic)) SGYYCTTTTMAGRRCS(SEQ ID NO: 16 according to formula (Ie)) DGNNNBNNTHVNNNCH(SEQ ID NO: 17 according to formula (Ie)) RGNNNYHBTHRDNNCY(SEQ ID NO: 18 according to formula (Ie)) RGNDBYHYTHRDHNCY(SEQ ID NO: 19 according to formula (If)) VGYYYTYHTHRVRRCB(SEQ ID NO: 20 according to formula (If)) SGYYCTTYTMAGRRCS(SEQ ID NO: 21 according to formula (If)) SGYYCTTTTMAGRRCS(SEQ ID NO: 22 according to formula (Ig)) GGYYCTTYTHAGRRCC(SEQ ID NO: 23 according to formula (Ig)) GGCYCTTYTMAGRGCC(SEQ ID NO: 24 according to formula (Ig)) GGCTCTTTTMAGRGCC(SEQ ID NO: 25 according to formula (Ih)) DGHYCTDYTHASRRCC(SEQ ID NO: 26 according to formula (Ih)) GGCYCTTTTHAGRGCC(SEQ ID NO: 27 according to formula (Ih)) GGCYCTTTTMAGRGCC

Furthermore in this context following histone stem-loop sequences (withstem bordering elements) as generated according to Example 1 accordingto one of specific formulae (II) or (IIa) to (IIh) are particularlypreferred:

(SEQ ID NO: 28 according to formula (IIc))H*H*HHVVGYYYYHHTHRVVRCBVHH*N*N*(SEQ ID NO: 29 according to formula (IIc))M*H*MHMSGYYYTTYTMARRRCSMCH*H*H*(SEQ ID NO: 30 according to formula (IIc))M*M*MMMSGYYCTTTTMAGRRCSACH*M*H*(SEQ ID NO: 31 according to formula (IIe))N*N*NNNDGNNNBNNTHVNNNCHNHN*N*N*(SEQ ID NO: 32 according to formula (IIe))N*N*HHNRGNNNYHBTHRDNNCYDHH*N*N*(SEQ ID NO: 33 according to formula (IIe))N*H*HHVRGNDBYHYTHRDHNCYRHH*H*H*(SEQ ID NO: 34 according to formula (IIf))H*H*MHMVGYYYTYHTHRVRRCBVMH*H*N*(SEQ ID NO: 35 according to formula (IIf))M*M*MMMSGYYCTTYTMAGRRCSMCH*H*H*(SEQ ID NO: 36 according to formula (IIf))M*M*MMMSGYYCTTTTMAGRRCSACH*M*H*(SEQ ID NO: 37 according to formula (IIg))H*H*MAMGGYYCTTYTHAGRRCCVHN*N*M*(SEQ ID NO: 38 according to formula (IIg))H*H*AAMGGCYCTTYTMAGRGCCVCH*H*M*(SEQ ID NO: 39 according to formula (IIg))M*M*AAMGGCTCTTTTMAGRGCCMCY*M*M*(SEQ ID NO: 40 according to formula (IIh))N*H*AAHDGHYCTDYTHASRRCCVHB*N*H*(SEQ ID NO: 41 according to formula (IIh))H*H*AAMGGCYCTTTTHAGRGCCVMY*N*M*(SEQ ID NO: 42 according to formula (IIh))H*M*AAAGGCYCTTTTMAGRGCCRMY*H*M*

According to a further preferred embodiment of the first inventiveaspect, the inventive nucleic acid sequence comprises or codes for atleast one histone stem-loop sequence showing at least about 80%,preferably at least about 85%, more preferably at least about 90%, oreven more preferably at least about 95%, sequence identity with the notto 100% conserved nucleotides in the histone stem-loop sequencesaccording to at least one of specific formulae (I) or (Ia) to (Ih) or(II) or (IIa) to (IIh) or with a naturally occurring histone stem-loopsequence.

In a preferred embodiment, the histone stem loop sequence does notcontain the loop sequence 5′-UUUC-3′. More specifically, the histonestem loop does not contain the stem1 sequence 5′-GGCUCU-3′ and/or thestem2 sequence 5′-AGAGCC-3′, respectively. In another preferredembodiment, the stem loop sequence does not contain the loop sequence5′-CCUGCCC-3′ or the loop sequence 5′-UGAAU-3′. More specifically, thestem loop does not contain the stem1 sequence 5′ -CCUGAGC-3′ or does notcontain the stem1 sequence 5′ -ACCUUUCUCCA-3′ (SEQ ID NO: 59) and/or thestem2 sequence 5′-GCUCAGG-3′ or 5′-UGGAGAAAGGU-3′ (SEQ ID NO: 60),respectively. Also, as far as the invention is not limited to histonestem loop sequences specifically, stem loop sequences are preferably notderived from a mammalian insulin receptor 3′-untranslated region. Also,preferably, the inventive nucleic acid may not contain histone stem loopprocessing signals, in particular not those derived from mouse histonegene H2A614 gene (H2kA614).

The inventive nucleic acid sequence according to the first aspect of thepresent invention may optionally comprise or code for a poly(A)sequence. When present, such a poly(A) sequence comprises a sequence ofabout 25 to about 400 adenosine nucleotides, preferably a sequence ofabout 30 or, more preferably, of about 50 to about 400 adenosinenucleotides, more preferably a sequence of about 50 to about 300adenosine nucleotides, even more preferably a sequence of about 50 toabout 250 adenosine nucleotides, most preferably a sequence of about 60to about 250 adenosine nucleotides. In this context the term “about”refers to a deviation of ±10% of the value(s) it is attached to.Accordingly, the poly(A) sequence contains at least 25 or more than 25,more preferably, at least 30, more preferably at least 50 adenosinenucleotides. Therefore, such a poly (A) sequence does typically notcontain less than 20 adenosine nucleotides. More particularly, it doesnot contain 10 and/or less than 10 adenosine nucleotides.

Preferably, the nucleic acid according of the present invention does notcontain one or two or at least one or all but one or all of thecomponents of the group consisting of: a sequence encoding a ribozyme(preferably a self-splicing ribozyme), a viral nucleic acid sequence, ahistone stem-loop processing signal, in particular a histone-stem loopprocessing sequence derived from mouse histone H2A614 gene, a Neo gene,an inactivated promoter sequence and an inactivated enhancer sequence.Even more preferably, the nucleic acid according to the invention doesnot contain a ribozyme, preferably a self-splicing ribozyme, and one ofthe group consisting of: a Neo gene, an inactivated promoter sequence,an inactivated enhancer sequence, a histone stem-loop processing signal,in particular a histone-stem loop processing sequence derived from mousehistone H2A614 gene. Accordingly, the nucleic acid may in a preferredmode neither contain a ribozyme, preferably a self-splicing ribozyme,nor a Neo gene or, alternatively, neither a ribozyme, preferably aself-splicing ribozyme, nor any resistance gene (e.g. usually appliedfor selection). In another preferred mode, the nucleic acid of theinvention may neither contain a ribozyme, preferably a self-splicingribozyme nor a histone stem-loop processing signal, in particular ahistone-stem loop processing sequence derived from mouse histone H2A614gene

Alternatively, according to the first aspect of the present invention,the inventive nucleic sequence optionally comprises a polyadenylationsignal which is defined herein as a signal which conveys polyadenylationto a (transcribed) mRNA by specific protein factors (e.g. cleavage andpolyadenylation specificity factor (CPSF), cleavage stimulation factor(CstF), cleavage factors I and II (CF I and CF II), poly(A) polymerase(PAP)). In this context a consensus polyadenylation signal is preferredcomprising the NN(U/T)ANA consensus sequence. In a particular preferredaspect the polyadenylation signal comprises one of the followingsequences: AA(U/T)AAA or A(U/T)(U/T)AAA (wherein uridine is usuallypresent in RNA and thymidine is usually present in DNA). In someembodiments, the polyadenylation signal used in the inventive nucleicacid does not correspond to the U3 snRNA, U5, the polyadenylationprocessing signal from human gene G-CSF, or the SV40 polyadenylationsignal sequences. In particular, the above polyadenylation signals arenot combined with any antibiotics resistance gene (or any otherreporter, marker or selection gene), in particular not with theresistance neo gene (neomycin phosphotransferase) (as the gene of thecoding region according to element (a) of the inventive nucleic acid.And, any of the above polyadenylation signals (which typically do notoccur in the inventive nucleic acid) are preferably not combined withthe histone stem loop or the histone stem loop processing signal frommouse histone gene H2A614 in an inventive nucleic acid.

The inventive nucleic acid sequence according to the first aspect of thepresent invention furthermore encodes a protein or a peptide, whichcomprises a therapeutic protein or a fragment, variant or derivativethereof.

Therapeutic proteins as defined herein are peptides or proteins whichare beneficial for the treatment of any inherited or acquired disease orwhich improves the condition of an individual. Particularly, therapeuticproteins plays a big role in the creation of therapeutic agents thatcould modify and repair genetic errors, destroy cancer cells or pathogeninfected cells, treat immune system disorders, treat metabolic orendocrine disorders, among other functions. For instance, Erythropoietin(EPO), a protein hormone can be utilized in treating patients witherythrocyte deficiency, which is a common cause of kidney complications.Furthermore adjuvant proteins, therapeutic antibodies are encompassed bytherapeutic proteins and also hormone replacement therapy which is e.g.used in the therapy of women in the menopause. In newer approachessomatic cells of a patient are used to reprogram them into pluripotentstem cells which replace the disputed stem cell therapy. Also theseproteins used for reprogramming of somatic cells or used fordifferentiating of stem cells are defined herein as therapeuticproteins. Furthermore therapeutic proteins may be used for otherpurposes e.g. wound healing, tissue regeneration, angiogenesis, etc.

Therefore therapeutic proteins can be used for various purposesincluding treatment of various diseases like e.g. infectious diseases,neoplasms (e.g. cancer or tumour diseases), diseases of the blood andblood-forming organs, endocrine, nutritional and metabolic diseases,diseases of the nervous system, diseases of the circulatory system,diseases of the respiratory system, diseases of the digestive system,diseases of the skin and subcutaneous tissue, diseases of themusculoskeletal system and connective tissue, and diseases of thegenitourinary system, independently if they are inherited or acquired.

In this context, particularly preferred therapeutic proteins which canbe used inter alia in the treatment of metabolic or endocrine disordersare selected from: Acid sphingomyelinase (Niemann-Pick disease),Adipotide (obesity), Agalsidase-beta (human galactosidase A) (Fabrydisease; prevents accumulation of lipids that could lead to renal andcardiovascular complications), Alglucosidase (Pompe disease (glycogenstorage disease type II)), alpha-galactosidase A (alpha-GAL A,Agalsidase alpha) (Fabry disease), alpha-glucosidase (Glycogen storagedisease (GSD), Morbus Pompe), alpha-L-iduronidase (mucopolysaccharidoses(MPS), Hurler syndrome, Scheie syndrome), alpha-N-acetylglucosaminidase(Sanfilippo syndrome), Amphiregulin (cancer, metabolic disorder),Angiopoietin ((Ang1, Ang2, Ang3, Ang4, ANGPTL2, ANGPTL3, ANGPTL4,ANGPTL5, ANGPTL6, ANGPTL7) (angiogenesis, stabilize vessels),Betacellulin (metabolic disorder), Beta-glucuronidase (Sly syndrome),Bone morphogenetic protein BMPs (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6,BMP7, BMP8a, BMP8b, BMP10, BMP15) (regenerative effect, bone-relatedconditions, chronic kidney disease (CKD)), CLN6 protein (CLN6disease—Atypical Late Infantile, Late Onset variant, Early Juvenile,Neuronal Ceroid Lipofuscinoses (NCL)), Epidermal growth factor (EGF)(wound healing, regulation of cell growth, proliferation, anddifferentiation), Epigen (metabolic disorder), Epiregulin (metabolicdisorder), Fibroblast Growth Factor (FGF, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, FGF-16, FGF-17, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22,FGF-23) (wound healing, angiogenesis, endocrine disorders, tissueregeneration), Galsulphase (Mucopolysaccharidosis VI), Ghrelin(irritable bowel syndrome (IBS), obesity, Prader-Willi syndrome, type IIdiabetes mellitus), Glucocerebrosidase (Gaucher's disease), GM-CSF(regenerative effect, production of white blood cells, cancer),Heparin-binding EGF-like growth factor (HB-EGF) (wound healing, cardiachypertrophy and heart development and function), Hepatocyte growthfactor HGF (regenerative effect, wound healing), Hepcidin (ironmetabolism disorders, Beta-thalassemia), Human albumin (Decreasedproduction of albumin (hypoproteinaemia), increased loss of albumin(nephrotic syndrome), hypovolaemia, hyperbilirubinaemia), Idursulphase(Iduronate-2-sulphatase) (Mucopolysaccharidosis II (Hunter syndrome)),Integrins αVβ3, αVβ5 and α5β1 (Bind matrix macromolecules andproteinases, angiogenesis), Iuduronate sulfatase (Hunter syndrome),Laronidase (Hurler and Hurler-Scheie forms of mucopolysaccharidosis I),N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase, Arylsulfatase A(ARSA), Arylsulfatase B (ARSB)) (arylsulfatase B deficiency,Maroteaux-Lamy syndrome, mucopolysaccharidosis VI),N-acetylglucosamine-6-sulfatase (Sanfilippo syndrome), Nerve growthfactor (NGF, Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3(NT-3), and Neurotrophin 4/5 (NT-4/5) (regenerative effect,cardiovascular diseases, coronary atherosclerosis, obesity, type 2diabetes, metabolic syndrome, acute coronary syndromes, dementia,depression, schizophrenia, autism, Rett syndrome, anorexia nervosa,bulimia nervosa, wound healing, skin ulcers, corneal ulcers, Alzheimer'sdisease), Neuregulin (NRG1, NRG2, NRG3, NRG4) (metabolic disorder,schizophrenia), Neuropilin (NRP-1, NRP-2) (angiogenesis, axon guidance,cell survival, migration), Obestatin (irritable bowel syndrome (IBS),obesity, Prader-Willi syndrome, type II diabetes mellitus), PlateletDerived Growth factor (PDGF (PDFF-A, PDGF-B, PDGF-C, PDGF-D)(regenerative effect, wound healing, disorder in angiogenesis,Arteriosclerosis, Fibrosis, cancer), TGF beta receptors (endoglin,TGF-beta 1 receptor, TGF-beta 2 receptor, TGF-beta 3 receptor) (renalfibrosis, kidney disease, diabetes, ultimately end-stage renal disease(ESRD), angiogenesis), Thrombopoietin (THPO) (Megakaryocyte growth anddevelopment factor (MGDF)) (platelets disorders, platelets for donation,recovery of platelet counts after myelosuppressive chemotherapy),Transforming Growth factor (TGF (TGF-a, TGF-beta (TGFbeta1, TGFbeta2,and TGFbeta3))) (regenerative effect, wound healing, immunity, cancer,heart disease, diabetes, Marfan syndrome, Loeys-Dietz syndrome), VEGF(VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PIGF) (regenerativeeffect, angiogenesis, wound healing, cancer, permeability), Nesiritide(Acute decompensated congestive heart failure), Trypsin (Decubitusulcer, varicose ulcer, debridement of eschar, dehiscent wound, sunburn,meconium ileus), adrenocorticotrophic hormone (ACTH) (“Addison'sdisease, Small cell carcinoma, Adrenoleukodystrophy, Congenital adrenalhyperplasia, Cushing's syndrome, Nelson's syndrome, Infantile spasms),Atrial-natriuretic peptide (ANP) (endocrine disorders), Cholecystokinin(diverse), Gastrin (hypogastrinemia), Leptin (Diabetes,hypertriglyceridemia, obesity), Oxytocin (stimulate breastfeeding,non-progression of parturition), Somatostatin (symptomatic treatment ofcarcinoid syndrome, acute variceal bleeding, and acromegaly, polycysticdiseases of the liver and kidney, acromegaly and symptoms caused byneuroendocrine tumors), Vasopressin (antidiuretic hormone) (diabetesinsipidus), Calcitonin (Postmenopausal osteoporosis, Hypercalcaemia,Paget's disease, Bone metastases, Phantom limb pain, Spinal Stenosis),Exenatide (Type 2 diabetes resistant to treatment with metformin and asulphonylurea), Growth hormone (GH), somatotropin (Growth failure due toGH deficiency or chronic renal insufficiency, Prader-Willi syndrome,Turner syndrome, AIDS wasting or cachexia with antiviral therapy),Insulin (Diabetes mellitus, diabetic ketoacidosis, hyperkalaemia),Insulin-like growth factor 1 IGF-1 (Growth failure in children with GHgene deletion or severe primary IGF1 deficiency, neurodegenerativedisease, cardiovascular diseases, heart failure), Mecasermin rinfabate,IGF-1 analog (Growth failure in children with GH gene deletion or severeprimary IGF 1 deficiency, neurodegenerative disease, cardiovasculardiseases, heart failure), Mecasermin, IGF-1 analog (Growth failure inchildren with GH gene deletion or severe primary IGF 1 deficiency,neurodegenerative disease, cardiovascular diseases, heart failure),Pegvisomant (Acromegaly), Pramlintide (Diabetes mellitus, in combinationwith insulin), Teriparatide (human parathyroid hormone residues 1-34)(Severe osteoporosis), Becaplermin (Debridement adjunct for diabeticulcers), Dibotermin-alpha (Bone morphogenetic protein 2) (Spinal fusionsurgery, bone injury repair), Histrelin acetate (gonadotropin releasinghormone; GnRH) (Precocious puberty), Octreotide (Acromegaly, symptomaticrelief of VIP-secreting adenoma and metastatic carcinoid tumours), andPalifermin (keratinocyte growth factor; KGF) (Severe oral mucositis inpatients undergoing chemotherapy, wound healing). (in brackets is theparticular disease for which the therapeutic protein is used in thetreatment). These and other proteins are understood to be therapeutic,as they are meant to treat the subject by replacing its defectiveendogenous production of a functional protein in sufficient amounts.Accordingly, such therapeutic proteins are typically mammalian, inparticular human proteins.

For the treatment of blood disorders, diseases of the circulatorysystem, diseases of the respiratory system, cancer or tumour diseases,infectious diseases or immunedeficiencies following therapeutic proteinsmay be used: Alteplase (tissue plasminogen activator; tPA) (Pulmonaryembolism, myocardial infarction, acute ischaemic stroke, occlusion ofcentral venous access devices), Anistreplase (Thrombolysis),Antithrombin III (AT-III) (Hereditary AT-III deficiency,Thromboembolism), Bivalirudin (Reduce blood-clotting risk in coronaryangioplasty and heparin-induced thrombocytopaenia), Darbepoetin-alpha(Treatment of anaemia in patients with chronic renal insufficiency andchronic renal failure (+/−dialysis)), Drotrecogin-alpha (activatedprotein C) (Severe sepsis with a high risk of death), Erythropoietin,Epoetin-alpha, erythropoetin, erthropoyetin (Anaemia of chronic disease,myleodysplasia, anaemia due to renal failure or chemotherapy,preoperative preparation), Factor IX (Haemophilia B), Factor VIIa(Haemorrhage in patients with haemophilia A or B and inhibitors tofactor VIII or factor IX), Factor VIII (Haemophilia A), Lepirudin(Heparin-induced thrombocytopaenia), Protein C concentrate (Venousthrombosis, Purpura fulminans), Reteplase (deletion mutein of tPA)(Management of acute myocardial infarction, improvement of ventricularfunction), Streptokinase (Acute evolving transmural myocardialinfarction, pulmonary embolism, deep vein thrombosis, arterialthrombosis or embolism, occlusion of arteriovenous cannula),Tenecteplase (Acute myocardial infarction), Urokinase (Pulmonaryembolism), Angiostatin (Cancer), Anti-CD22 immunotoxin (Relapsed CD33+acute myeloid leukaemia), Denileukin diftitox (Cutaneous T-cell lymphoma(CTCL)), Immunocyanin (bladder and prostate cancer), MPS(Metallopanstimulin) (Cancer), Aflibercept (Non-small cell lung cancer(NSCLC), metastatic colorectal cancer (mCRC), hormone-refractorymetastatic prostate cancer, wet macular degeneration), Endostatin(Cancer, inflammatory diseases like rheumatoid arthritis as well asCrohn's disease, diabetic retinopathy, psoriasis, and endometriosis),Collagenase (Debridement of chronic dermal ulcers and severely burnedareas, Dupuytren's contracture, Peyronie's disease), Humandeoxy-ribonuclease I, dornase (Cystic fibrosis; decreases respiratorytract infections in selected patients with FVC greater than 40% ofpredicted), Hyaluronidase (Used as an adjuvant to increase theabsorption and dispersion of injected drugs, particularly anaestheticsin ophthalmic surgery and certain imaging agents), Papain (Debridementof necrotic tissue or liquefication of slough in acute and chroniclesions, such as pressure ulcers, varicose and diabetic ulcers, burns,postoperative wounds, pilonidal cyst wounds, carbuncles, and otherwounds), L-Asparaginase (Acute lymphocytic leukaemia, which requiresexogenous asparagine for proliferation), Peg-asparaginase (Acutelymphocytic leukaemia, which requires exogenous asparagine forproliferation), Rasburicase (Paediatric patients with leukaemia,lymphoma, and solid tumours who are undergoing anticancer therapy thatmay cause tumour lysis syndrome), Human chorionic gonadotropin (HCG)(Assisted reproduction), Human follicle-stimulating hormone (FSH)(Assisted reproduction), Lutropin-alpha (Infertility with luteinizinghormone deficiency), Prolactin (Hypoprolactinemia, serum prolactindeficiency, ovarian dysfunction in women, anxiety, arteriogenic erectiledysfunction, premature ejaculation, oligozoospermia, asthenospermia,hypofunction of seminal vesicles, hypoandrogenism in men),alpha-1-Proteinase inhibitor (Congenital antitrypsin deficiency),Lactase (Gas, bloating, cramps and diarrhoea due to inability to digestlactose), Pancreatic enzymes (lipase, amylase, protease) (Cysticfibrosis, chronic pancreatitis, pancreatic insufficiency, post-BillrothII gastric bypass surgery, pancreatic duct obstruction, steatorrhoea,poor digestion, gas, bloating), Adenosine deaminase (pegademase bovine,PEG-ADA) (Severe combined immunodeficiency disease due to adenosinedeaminase deficiency), Abatacept (Rheumatoid arthritis (especially whenrefractory to TNFa inhibition)), Alefacept (Plaque Psoriasis), Anakinra(Rheumatoid arthritis), Etanercept (Rheumatoid arthritis,polyarticular-course juvenile rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, plaque psoriasis, ankylosing spondylitis),Interleukin-1 (IL-1) receptor antagonist, Anakinra (inflammation andcartilage degradation associated with rheumatoid arthritis), Thymulin(neurodegenerative diseases, rheumatism, anorexia nervosa), TNF-alphaantagonist (autoimmune disorders such as rheumatoid arthritis,ankylosing spondylitis, Crohn's disease, psoriasis, hidradenitissuppurativa, refractory asthma), Enfuvirtide (HIV-1 infection), andThymosin α1 (Hepatitis B and C). (in brackets is the particular diseasefor which the therapeutic protein is used in the treatment)

Furthermore adjuvant or immunostimulating proteins are also encompassedin the term therapeutic proteins. Adjuvant or immunostimulating proteinsmay be used in this context to induce, alter or improve an immuneresponse in an individual to treat a particular disease or to amelioratethe condition of the individual.

In this context adjuvant proteins may be selected from mammalian, inparticular human adjuvant proteins, which typically comprise any humanprotein or peptide, which is capable of eliciting an innate immuneresponse (in a mammal), e.g. as a reaction of the binding of anexogenous TLR ligand to a TLR. More preferably, human adjuvant proteinsare selected from the group consisting of proteins which are componentsand ligands of the signalling networks of the pattern recognitionreceptors including TLR, NLR and RLH, including TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NOD1, NOD2, NOD3, NOD4,NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7, NALP7,NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14,1 IPAF, NAIP,CIITA, RIG-I, MDA5 and LGP2, the signal transducers of TLR signalingincluding adaptor proteins including e.g. Trif and Cardif; components ofthe Small-GTPases signalling (RhoA, Ras, Rac1, Cdc42, Rab etc.),components of the PIP signalling (PI3K, Src-Kinases, etc.), componentsof the MyD88-dependent signalling (MyD88, IRAK1, IRAK2, IRAK4, TIRAP,TRAF6 etc.), components of the MyD88-independent signalling (TICAM1,TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1 etc.); the activated kinasesincluding e.g. Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2,GSK3, PKC kinases, PKD kinases, GSK3 kinases, JNK, p38MAPK, TAK1, IKK,and TAK1; the activated transcription factors including e.g. NF-κB,c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7.

Mammalian, in particular human adjuvant proteins may furthermore beselected from the group consisting of heat shock proteins, such asHSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIIIrepeat extra domain A of fibronectin; or components of the complementsystem including C1q, MBL, C1r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b,C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR,C1INH, C4bp, MCP, DAF, H, I, P and CD59, or induced target genesincluding e.g. Beta-Defensin, cell surface proteins; or human adjuvantproteins including trif, flt-3 ligand, Gp96 or fibronectin, etc., or anyspecies homolog of any of the above human adjuvant proteins.

Mammalian, in particular human adjuvant proteins may furthermorecomprise cytokines which induce or enhance an innate immune response,including IL-1 alpha, IL1 beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12,IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNFalpha, IFNalpha,IFNbeta, IFNgamma, GM-CSF, G-CSF, M-CSF; chemokines including IL-8,IP-10, MCP-1, MIP-lalpha, RANTES, Eotaxin, CCL21; cytokines which arereleased from macrophages, including IL-1, IL-6, IL-8, IL-12 andTNF-alpha; as well as IL-1R1 and IL-1 alpha.

Therapeutic proteins for the treatment of blood disorders, diseases ofthe circulatory system, diseases of the respiratory system, cancer ortumour diseases, infectious diseases or immunedeficiencies or adjuvantproteins are typically proteins of mammalian origin, preferably of humanorigin, depending on which animal shall be treated. A human is e.g.preferably treated by a therapeutic protein of human origin.

Pathogenic adjuvant proteins, typically comprise any pathogenic adjuvantprotein, which is capable of eliciting an innate immune response (in amammal), more preferably selected from pathogenic adjuvant proteinsderived from bacteria, protozoa, viruses, or fungi, etc., e.g.,bacterial (adjuvant) proteins, protozoan (adjuvant) proteins (e.g.profilin—like protein of Toxoplasma gondii), viral (adjuvant) proteins,or fungal (adjuvant) proteins, etc.

Particularly, bacterial (adjuvant) proteins may be selected from thegroup consisting of bacterial heat shock proteins or chaperons,including Hsp60, Hsp70, Hsp90, Hsp100; OmpA (Outer membrane protein)from gram-negative bacteria; bacterial porins, including OmpF; bacterialtoxins, including pertussis toxin (PT) from Bordetella pertussis,pertussis adenylate cyclase toxin CyaA and CyaC from Bordetellapertussis, PT-9K/129G mutant from pertussis toxin, pertussis adenylatecyclase toxin CyaA and CyaC from Bordetella pertussis, tetanus toxin,cholera toxin (CT), cholera toxin B-subunit, CTK63 mutant from choleratoxin, CTE112K mutant from CT, Escherichia coli heat-labile enterotoxin(LT), B subunit from heat-labile enterotoxin (LTB) Escherichia coliheat-labile enterotoxin mutants with reduced toxicity, including LTK63,LTR72; phenol-soluble modulin; neutrophil-activating protein (HP-NAP)from Helicobacter pylori; Surfactant protein D; Outer surface protein Alipoprotein from Borrelia burgdorferi, Ag38 (38 kDa antigen) fromMycobacterium tuberculosis; proteins from bacterial fimbriae;Enterotoxin CT of Vibrio cholerae, Pilin from pili from gram negativebacteria, and Surfactant protein A; etc., or any species homolog of anyof the above bacterial (adjuvant) proteins.

Bacterial (adjuvant) proteins may also comprise bacterial flagellins. Inthe context of the present invention, bacterial flagellins may beselected from flagellins from organisms including, without being limitedthereto, Agrobacterium, Aquifex, Azospirillum, Bacillus, Bartonella,Bordetella, Borrelia, Burkholderia, Campylobacter, Caulobacte,Clostridium, Escherichia, Helicobacter, Lachnospiraceae, Legionella,Listeria, Proteus, Pseudomonas, Rhizobium, Rhodobacter, Roseburia,Salmonella, Serpulina, Serratia, Shigella, Treponema, Vibrio, Wolinella,Yersinia, more preferably from flagellins from the species including,without being limited thereto, Agrobacterium tumefaciens, Aquifexpyrophilus, Azospirillum brasilense, Bacillus subtilis, Bacillusthuringiensis, Bartonella bacilliformis, Bordetella bronchiseptica,Borrelia burgdorferi, Burkholderia cepacia, Campylobacter jejuni,Caulobacter crescentus, Clostridium botulinum strain Bennett clone 1,Escherichia coli, Helicobacter pylori, Lachnospiraceae bacterium,Legionella pneumophila, Listeria monocytogenes, Proteus mirabilis,Pseudomonas aeroguinosa, Pseudomonas syringae, Rhizobium meliloti,Rhodobacter sphaeroides, Roseburia cecicola, Roseburis hominis,Salmonella typhimurium, Salmonella bongori, Salmonella typhi, Salmonellaenteritidis, Serpulina hyodysenteriae, Serratia marcescens, Shigellaboydii, Treponema phagedenis, Vibrio alginolyticus, Vibrio cholerae,Vibrio parahaemolyticus, Wolinella succinogenes and Yersiniaenterocolitica.

Protozoan (adjuvant) proteins are a further example of pathogenicadjuvant proteins. Protozoan (adjuvant) proteins may be selected in thiscontext from any protozoan protein showing adjuvant character, morepreferably, from the group consisting of, without being limited thereto,Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii, Protozoanheat shock proteins, LeIF from Leishmania spp., profiling-like proteinfrom Toxoplasma gondii, etc.

Viral (adjuvant) proteins are another example of pathogenic adjuvantproteins. In this context, viral (adjuvant) proteins may be selectedfrom any viral protein showing adjuvant character, more preferably, fromthe group consisting of, without being limited thereto, RespiratorySyncytial Virus fusion glycoprotein (F-protein), envelope protein fromMMT virus, mouse leukemia virus protein, Hemagglutinin protein ofwild-type measles virus, etc.

Fungal (adjuvant) proteins are even a further example of pathogenicadjuvant proteins. In the context of the present invention, fungal(adjuvant) proteins may be selected from any fungal protein showingadjuvant character, more preferably, from the group consisting of,fungal immunomodulatory protein (FIP; LZ-8), etc.

Finally, adjuvant proteins may furthermore be selected from the groupconsisting of, Keyhole limpet hemocyanin (KLH), OspA, etc.

In a further embodiment therapeutic proteins may be used for hormonereplacement therapy, particularly for the therapy of women in themenopause. These therapeutic proteins are preferably selected fromoestrogens, progesterone or progestins, and sometimes testosterone.

Furthermore, therapeutic proteins may be used for reprogramming ofsomatic cells into pluri- or omnipotent stem cells. For this purposeseveral factors are described, particularly Oct-3/4, Sox gene family(Sox1, Sox2, Sox3, and Sox15), Klf family (Klf1, Klf2, Klf4, and Klf5),Myc family (c-myc, L-myc, and N-myc), Nanog, and LIN28.

As mentioned above, also therapeutic antibodies are defined herein astherapeutic proteins. These therapeutic antibodies are preferablyselected from antibodies which are used inter alia for the treatment ofcancer or tumour diseases, e.g. 131I-tositumomab (Follicular lymphoma, Bcell lymphomas, leukemias), 3F8 (Neuroblastoma), 8H9, Abagovomab(Ovarian cancer), Adecatumumab (Prostate and breast cancer), Afutuzumab(Lymphoma), Alacizumab pegol, Alemtuzumab (B-cell chronic lymphocyticleukaemia, T-cell-Lymphoma), Amatuximab, AME-133v (Follicular lymphoma,cancer), AMG 102 (Advanced Renal Cell Carcinoma), Anatumomab mafenatox(Non-small cell lung carcinoma), Apolizumab (Solid Tumors, Leukemia,Non-Hodgkin-Lymphoma, Lymphoma), Bavituximab (Cancer, viral infections),Bectumomab (Non-Hodgkin's lymphoma), Belimumab (Non-Hodgkin lymphoma),Bevacizumab (Colon Cancer, Breast Cancer, Brain and Central NervousSystem Tumors, Lung Cancer, Hepatocellular Carcinoma, Kidney Cancer,Breast Cancer, Pancreatic Cancer, Bladder Cancer, Sarcoma, Melanoma,Esophageal Cancer; Stomach Cancer, Metastatic Renal Cell Carcinoma;Kidney Cancer, Glioblastoma, Liver Cancer, Proliferative DiabeticRetinopathy, Macular Degeneration), Bivatuzumab mertansine (Squamouscell carcinoma), Blinatumomab, Brentuximab vedotin (Hematologiccancers), Cantuzumab (Colon Cancer, Gastric Cancer, Pancreatic Cancer,NSCLC), Cantuzumab mertansine (Colorectal cancer), Cantuzumab ravtansine(Cancers), Capromab pendetide (Prostate cancer), Carlumab, Catumaxomab(Ovarian Cancer, Fallopian Tube Neoplasms, Peritoneal Neoplasms),Cetuximab (Metastatic colorectal cancer and head and neck cancer),Citatuzumab bogatox (Ovarian cancer and other solid tumors), Cixutumumab(Solid tumors), Clivatuzumab tetraxetan (Pancreatic cancer), CNTO 328(B-Cell Non-Hodgkin's Lymphoma, Multiple Myeloma, Castleman's Disease,ovarian cancer), CNTO 95 (Melanoma), Conatumumab, Dacetuzumab(Hematologic cancers), Dalotuzumab, Denosumab (Myeloma, Giant Cell Tumorof Bone, Breast Cancer, Prostate Cancer, Osteoporosis), Detumomab(Lymphoma), Drozitumab, Ecromeximab (Malignant melanoma), Edrecolomab(Colorectal carcinoma), Elotuzumab (Multiple myeloma), Elsilimomab,Enavatuzumab, Ensituximab, Epratuzumab (Autoimmune diseases, SystemicLupus Erythematosus, Non-Hodgkin-Lymphoma, Leukemia), Ertumaxomab(Breast cancer), Ertumaxomab (Breast Cancer), Etaracizumab (Melanoma,prostate cancer, ovarian cancer), Farletuzumab (Ovarian cancer), FBTA05(Chronic lymphocytic leukaemia), Ficlatuzumab (Cancer), Figitumumab(Adrenocortical carcinoma, non-small cell lung carcinoma), Flanvotumab(Melanoma), Galiximab (B-cell lymphoma), Galiximab(Non-Hodgkin-Lymphoma), Ganitumab, GC1008 (Advanced Renal CellCarcinoma; Malignant Melanoma, Pulmonary Fibrosis), Gemtuzumab(Leukemia), Gemtuzumab ozogamicin (Acute myelogenous leukemia),Girentuximab (Clear cell renal cell carcinoma), Glembatumumab vedotin(Melanoma, breast cancer), GS6624 (Idiopathic pulmonary fibrosis andsolid tumors), HuC242-DM4 (Colon Cancer, Gastric Cancer, PancreaticCancer), HuHMFG1 (Breast Cancer), HuN901-DM1 (Myeloma), Ibritumomab(Relapsed or refractory low-grade, follicular, or transformed B-cellnon-Hodgkin's lymphoma (NHL)), Icrucumab, ID09C3 (Non-Hodgkin-Lymphoma),Indatuximab ravtansine, Inotuzumab ozogamicin, Intetumumab (Solid tumors(Prostate cancer, melanoma)), Ipilimumab (Sarcoma, Melanoma, Lungcancer, Ovarian Cancer leucemia, Lymphoma, Brain and Central NervousSystem Tumors, Testicular Cancer, Prostate Cancer, Pancreatic Cancer,Breast Cancer), Iratumumab (Hodgkin's lymphoma), Labetuzumab (Colorectalcancer), Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab(Multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma),Lumiliximab (Chronic lymphocytic leukemia), Mapatumumab (Colon Cancer,Myeloma), Matuzumab (Lung Cancer, Cervical Cancer, Esophageal Cancer),MDX-060 (Hodgkin-Lymphoma, Lymphoma), MEDI 522 (Solid Tumors, Leukemia,Lymphoma, Small Intestine Cancer, Melanoma), Mitumomab (Small cell lungcarcinoma), Mogamulizumab, MORab-003 (Ovarian Cancer, Fallopian TubeCancer, Peritoneal Cancer), MORab-009 (Pancreatic Cancer, Mesothelioma,Ovarian Cancer, Non-Small Cell Lung Cancer, Fallopian Tube Cancer,Peritoneal Cavity Cancer), Moxetumomab pasudotox, MT103(Non-Hodgkin-Lymphoma), Nacolomab tafenatox (Colorectal cancer),Naptumomab estafenatox (Non-small cell lung carcinoma, renal cellcarcinoma), Narnatumab, Necitumumab (Non-small cell lung carcinoma),Nimotuzumab (Squamous cell carcinoma, head and neck cancer,nasopharyngeal cancer, glioma), Nimotuzumab (Squamous cell carcinomas,Glioma, Solid Tumors, Lung Cancer), Olaratumab, Onartuzumab (Cancer),Oportuzumab monatox, Oregovomab (Ovarian cancer), Oregovomab (OvarianCancer, Fallopian Tube Cancer, Peritoneal Cavity Cancer), PAM4(Pancreatic Cancer), Panitumumab (Colon Cancer, Lung Cancer, BreastCancer; Bladder Cancer; Ovarian Cancer), Patritumab, Pemtumomab,Pertuzumab (Breast Cancer, Ovarian Cancer, Lung Cancer, ProstateCancer), Pritumumab (Brain cancer), Racotumomab, Radretumab, Ramucirumab(Solid tumors), Rilotumumab (Solid tumors), Rituximab (Urticaria,Rheumatoid Arthritis, Ulcerative Colitis, Chronic Focal Encephalitis,Non-Hodgkin-Lymphoma, Lymphoma, Chronic Lymphocytic Leukemia),Robatumumab, Samalizumab, SGN-30 (Hodgkin-Lymphoma, Lymphoma), SGN-40(Non-Hodgkin-Lymphoma, Myeloma, Leukemia, Chronic Lymphocytic Leukemia),Sibrotuzumab, Siltuximab, Tabalumab (B-cell cancers), Tacatuzumabtetraxetan, Taplitumomab paptox, Tenatumomab, Teprotumumab (Hematologictumors), TGN1412 (Chronic lymphocytic leukemia, rheumatoid arthritis),Ticilimumab, Tigatuzumab, TNX-650 (Hodgkin's lymphoma), Tositumomab(Follicular lymphoma, B cell lymphomas, Leukemias, Myeloma), Trastuzumab(Breast Cancer, Endometrial Cancer, Solid Tumors), TRB S07 (Melanoma),Tremelimumab, TRU-016 (Chronic lymphocytic leukemia), TRU-016(Non-Hodgkin lymphoma), Tucotuzumab celmoleukin, Ublituximab, Urelumab,Veltuzumab (Non-Hodgkin's lymphoma), Veltuzumab (IMMU-106)(Non-Hodgkin's lymphoma), Volociximab (Renal Cell Carcinoma, PancreaticCancer, Melanoma), Votumumab (Colorectal tumors), WX-G250 (Renal CellCarcinoma), Zalutumumab (Head and Neck Cancer, Squamous Cell Cancer),and Zanolimumab (T-Cell-Lymphoma);

antibodies which are used inter alia for the treatment of immunedisorders, e.g. Efalizumab (Psoriasis), Epratuzumab (Autoimmunediseases, Systemic Lupus Erythematosus, Non-Hodgkin-Lymphoma, Leukemia),Etrolizumab (inflammatory bowel disease), Fontolizumab (Crohn'sdisease), Ixekizumab (autoimmune diseases), Mepolizumab(Hypereosinophilie-Syndrom, Asthma, Eosinophilic Gastroenteritis,Churg-Strauss Syndrome, Eosinophilic Esophagitis), Milatuzumab (multiplemyeloma and other hematological malignancies), Pooled immunoglobulins(Primary immunodeficiencies), Priliximab (Crohn's disease, multiplesclerosis), Rituximab (Urticaria, Rheumatoid Arthritis, UlcerativeColitis, Chronic Focal Encephalitis, Non-Hodgkin-Lymphoma, Lymphoma,Chronic Lymphocytic Leukemia), Rontalizumab (systemic lupuserythematosus), Ruplizumab (rheumatic diseases), Sarilumab (rheumatoidarthritis, ankylosing spondylitis), Vedolizumab (Crohn's disease,ulcerative colitis), Visilizumab (Crohn's disease, ulcerative colitis),Reslizumab (inflammations of the airways, skin and gastrointestinaltract), Adalimumab (Rheumatoid arthritis, Crohn's disease, Ankylosingspondylitis, Psoriatic arthritis), Aselizumab (severely injuredpatients), Atinumab (treatment of neurologic systems), Atlizumab(rheumatoid arthritis, systemic juvenile idiopathic arthritis),Bertilimumab (severe allergic disorders), Besilesomab (inflammatorylesions and metastases), BMS-945429, ALD518 (cancer and rheumatoidarthritis), Briakinumab (psoriasis, rheumatoid arthritis, inflammatorybowel diseases, multiple sclerosis), Brodalumab (inflammatory diseases),Canakinumab (rheumatoid arthritis), Canakinumab (cryopyrin-associatedperiodic syndromes (CAPS), rheumatoid arthritis, chronic obstructivepulmonary disease), Certolizumab pegol (Crohn's disease), Erlizumab(heart attack, stroke, traumatic shock), Fezakinumab (rheumatoidarthritis, psoriasis), Golimumab (rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis), Gomiliximab (allergic asthma),Infliximab (Rheumatoid arthritis, Crohn's disease, ankylosingspondylitis, psoriatic arthritis, plaque psoriasis, Morbus Bechterew,Colitis ulcerosa), Mavrilimumab (rheumatoid arthritis), Natalizumab(Multiple sclerosis), Ocrelizumab (multiple sclerosis, rheumatoidarthritis, lupus erythematosus, hematological cancer), Odulimomab(prevention of organ transplant rejections, immunological diseases),Ofatumumab (Chronic lymphocytic leukemia, follicular non-Hodgkin'slymphoma, B cell lymphoma, rheumatoid arthritis, relapsing remittingmultiple sclerosis, Lymphoma, B-Cell Chronic Lymphocytic Leukemia),Ozoralizumab (inflammation), Pexelizumab (reduction of side effects ofcardiac surgery), Rovelizumab (haemorrhagic shock), SBI-087 (Rheumatoidarthritis), SBI-087 (Systemic lupus erythematosus), Secukinumab(uveitis, rheumatoid arthritis psoriasis), Sirukumab (rheumatoidarthritis), Talizumab (allergic reaction), Tocilizumab (rheumatoidarthritis, systemic juvenile idiopathic arthritis, Castleman's disease),Toralizumab (rheumatoid arthritis, lupus nephritis), TRU-015 (Rheumatoidarthritis), TRU-016 (Autoimmune disease and inflammation), Ustekinumab(multiple sclerosis, psoriasis, psoriatic arthritis), Ustekinumab(IL-12/IL-23 blocker) (Plaque-Psoriasis, psoriatic arthritis, multiplesclerosis, sarcoidosis, the latter versus), Vepalimomab (inflammation),Zolimomab aritox (systemic lupus erythematosus, graft-versus-hostdisease), Sifalimumab (SLE, dermatomyositis, polymyositis), Lumiliximab(Allergies), and Rho(D) Immune Globulin (Rhesus disease); or areselected from antibodies used for the treatment of infectious diseases,e.g. Afelimomab (sepsis), CR6261 (infectious disease/influenza A),Edobacomab (sepsis caused by gram-negative bacteria), Efungumab(invasive Candida infection), Exbivirumab (hepatitis B), Felvizumab(respiratory syncytial virus infection), Foravirumab (rabies(prophylaxis)), Ibalizumab (HIV infection), Libivirumab (hepatitis B),Motavizumab (respiratory syncytial virus (prevention)), Nebacumab(sepsis), Tuvirumab (chronic hepatitis B), Urtoxazumab (diarrhoea causedby E. coli), Bavituximab (diverse viral infections), Pagibaximab (sepsis(e.g. Staphylococcus)), Palivizumab (prevention of respiratory syncytialvirus infection in high-risk paediatric patients), Panobacumab(Pseudomonas aeruginosa infection), PRO 140 (HIV infection), Rafivirumab(rabies (prophylaxis)), Raxibacumab (anthrax (prophylaxis andtreatment)), Regavirumab (cytomegalovirus infection), Sevirumab(cytomegalovirus infection), Suvizumab (viral infections), andTefibazumab (Staphylococcus aureus infection);

antibodies which are used inter alia for the treatment of blooddisorders, e.g. Abciximab (percutaneous coronary intervention),Atorolimumab (hemolytic disease of the newborn), Eculizumab (Paroxysmalnocturnal haemoglobinuria), Mepolizumab (Hypereosinophilie-Syndrom,Asthma, Eosinophilic Gastroenteritis, Churg-Strauss Syndrome,Eosinophilic Esophagitis), and Milatuzumab (multiple myeloma and otherhematological malignancies);

antibodies which are used inter alia for immunoregulation, e.g.Antithymocyte globulin (Acute kidney transplant rejection, aplasticanaemia), Basiliximab (Prophylaxis against allograft rejection in renaltransplant patients receiving an immunosuppressive regimen includingcyclosporine and corticosteroids), Cedelizumab (prevention of organtransplant rejections, treatment of autoimmune diseases), Daclizumab(Prophylaxis against acute allograft rejection in patients receivingrenal transplants, Multiple Sclerosis), Gavilimomab (graft versus hostdisease), Inolimomab (graft versus host disease), Muromonab-CD3(prevention of organ transplant rejections), Muromonab-CD3 (Acute renalallograft rejection or steroid-resistant cardiac or hepatic allograftrejection), Odulimomab (prevention of organ transplant rejections,immunological diseases), and Siplizumab (psoriasis, graft-versus-hostdisease (prevention));

antibodies used for the treatment of diabetes, e.g. Gevokizumab(diabetes), Otelixizumab (diabetes mellitus type 1), and Teplizumab(diabetes mellitus type 1);

antibodies which are used for the treatment of the Alzheimer's disease,e.g. Bapineuzumab, Crenezumab, Gantenerumab, Ponezumab, R1450, andSolanezumab;

antibodies which are used for the treatment of asthma, e.g.Benralizumab, Enokizumab, Keliximab, Lebrikizumab, Omalizumab, Oxelumab,Pascolizumab, and Tralokinumab;

and antibodies which are used for the treatment of diverse disorders,e.g. Blosozumab (osteoporosis), CaroRx (Tooth decay), Fresolimumab(idiopathic pulmonary fibrosis, focal segmental glomerulosclerosis,cancer), Fulranumab (pain), Romosozumab (osteoporosis), Stamulumab(muscular dystrophy), Tanezumab (pain), and Ranibizumab (Neovascularage-related macular degeneration).

The coding region of the inventive nucleic acid according to the firstaspect of the present invention may occur as a mono-, di-, or evenmulticistronic nucleic acid, i.e. a nucleic acid which carries thecoding sequences of one, two or more proteins or peptides. Such codingsequences in di-, or even multicistronic nucleic acids may be separatedby at least one internal ribosome entry site (IRES) sequence, e.g. asdescribed herein or by signal peptides which induce the cleavage of theresulting polypeptide which comprises several proteins or peptides.

According to the first aspect of the present invention, the inventivenucleic acid sequence comprises a coding region, encoding a peptide orprotein which comprises a therapeutic protein or a fragment, variant orderivative thereof. Preferably, the encoded therapeutic protein is nohistone protein. In the context of the present invention such a histoneprotein is typically a strongly alkaline protein found in eukaryoticcell nuclei, which package and order the DNA into structural unitscalled nucleosomes. Histone proteins are the chief protein components ofchromatin, act as spools around which DNA winds, and play a role in generegulation. Without histones, the unwound DNA in chromosomes would bevery long (a length to width ratio of more than 10 million to one inhuman DNA). For example, each human cell has about 1.8 meters of DNA,but wound on the histones it has about 90 millimeters of chromatin,which, when duplicated and condensed during mitosis, result in about 120micrometers of chromosomes. More preferably, in the context of thepresent invention such a histone protein is typically defined as ahighly conserved protein selected from one of the following five majorclasses of histones: H1/H5, H2A, H2B, H3, and H4″, preferably selectedfrom mammalian histone, more preferably from human histones or histoneproteins. Such histones or histone proteins are typically organised intotwo super-classes defined as core histones, comprising histones H2A,H2B, H3 and H4, and linker histones, comprising histones H1 and H5.

In this context, linker histones, preferably excluded from the scope ofprotection of the pending invention, preferably mammalian linkerhistones, more preferably human linker histones, are typically selectedfrom H1, including H1F, particularly including H1F0, H1FNT, H1FOO, H1FX,and H1H1, particularly including HIST1H1A, HIST1H1B, HIST1H1C, HIST1H1D,HIST1H1E, HIST1H1T; and

Furthermore, core histones, preferably excluded from the scope ofprotection of the pending invention, preferably mammalian core histones,more preferably human core histones, are typically selected from H2A,including H2AF, particularly including H2AFB1, H2AFB2, H2AFB3, H2AFJ,H2AFV, H2AFX, H2AFY, H2AFY2, H2AFZ, and H2A1, particularly includingHIST1H2AA, HIST1H2AB, HIST1H2AC, HIST1H2AD, HIST1H2AE, HIST1H2AG,HIST1H2AI, HIST1H2AJ, HIST1H2AK, HIST1H2AL, HIST1H2AM, and H2A2,particularly including HIST2H2AA3, HIST2H2AC; H2B, including H2BF,particularly including H2BFM, H2BFO, H2BFS, H2BFWT H2B1, particularlyincluding HIST1H2BA, HIST1H2BB, HIST1H2BC, HIST1H2BD, HIST1H2BE,HIST1H2BF, HIST1H2BG, HIST1H2BH, HIST1H2BI, HIST1H2BJ, HIST1H2BK,HIST1H2BL, HIST1H2BM, HIST1H2BN, HIST1H2BO, and H2B2, particularlyincluding HIST2H2BE; H3, including H3A1, particularly includingHIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G,HIST1H3H, HIST1H3I, HIST1H3J, and H3A2, particularly including HIST2H3C,and H3A3, particularly including HIST3H3; H4, including H41,particularly including HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E,HIST1H4F, HIST1H4G, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4L,and H44, particularly including HIST4H4, and H5.

According to the first aspect of the present invention, the inventivenucleic acid sequence comprises a coding region, encoding a peptide orprotein which comprises a therapeutic protein or a fragment, variant orderivative thereof. Preferably, the encoded therapeutic protein is noreporter protein (e.g. Luciferase, Green Fluorescent Protein (GFP),Enhanced Green Fluorescent Protein (EGFP), β-Galactosidase) and nomarker or selection protein (e.g. alpha-Globin, Galactokinase andXanthine:guanine phosphoribosyl transferase (GPT)). Preferably, thenucleic acid sequence of the invention does not contain a (bacterial)antibiotics resistance gene, in particular not a neo gene sequence(Neomycin resistance gene) or CAT gene sequence (chloramphenicol acetyltransferase, chloramphenicol resistance gene).

The inventive nucleic acid as define above, comprises or codes for a) acoding region, encoding a peptide or protein which comprises atherapeutic protein or a fragment, variant or derivative thereof; b) atleast one histone stem-loop, and c) a poly(A) sequence orpolyadenylation signal;

preferably for increasing the expression of said encoded peptide orprotein, wherein the encoded peptide or protein is preferably no histoneprotein, no reporter protein and/or no marker or selection protein, asdefined above. The elements b) to c) of the inventive nucleic acid mayoccur in the inventive nucleic acid in any order, i.e. the elements a),b) and c) may occur in the order a), b) and c) or a), c) and b) from 5′to 3′ direction in the inventive nucleic acid sequence, wherein furtherelements as described herein, may also be contained, such as a 5′-CAPstructure, a poly(C) sequence, stabilization sequences, IRES sequences,etc. Each of the elements a) to c) of the inventive nucleic acid,particularly a) in di- or multicistronic constructs and/or each of theelements b) and c), more preferably element b) may also be repeated atleast once, preferably twice or more in the inventive nucleic acid. Asan example, the inventive nucleic acid may show its sequence elementsa), b) and optionally c) in e.g. the following order:

5′-coding region-histone stem-loop-poly(A)  sequence-3′; or5′-coding region-histone stem-loop-polyadenylation  signal-3′; or5′-coding region-poly(A) sequence-histone stem-  loop-3′; or5′-coding region-polyadenylation signal-histone stem-loop-3′;  or5′-coding region-coding region-histone stem-loop- polyadenylation signal-3′; or5′-coding region-histone stem-loop-histone stem- loop-poly(A) sequence-3′; or5′-coding region-histone stem-loop-histone stem-loop-polyadenylation signal-3′; etc.

In this context it is particularly preferred that the inventive nucleicacid sequence comprises or codes for a) a coding region, encoding apeptide or protein which comprises a therapeutic protein or fragment,variant or derivative thereof; b) at least one histone stem-loop, and c)a poly(A) sequence or polyadenylation sequence; preferably forincreasing the expression level of said encoded peptide or protein,wherein the encoded protein is preferably no histone protein, noreporter protein (e.g. Luciferase, GFP, EGFP, β-Galactosidase,particularly EGFP) and/or no marker or selection protein (e.g.alpha-Globin, Galactokinase and Xanthine:Guanine phosphoribosyltransferase (GPT)).

In a further preferred embodiment of the first aspect the inventivenucleic acid sequence as defined herein may also occur in the form of amodified nucleic acid.

In this context, the inventive nucleic acid sequence as defined hereinmay be modified to provide a “stabilized nucleic acid”, preferably astabilized RNA, more preferably an RNA that is essentially resistant toin vivo degradation (e.g. by an exo- or endo-nuclease). A stabilizednucleic acid may e.g. be obtained by modification of the G/C content ofthe coding region of the inventive nucleic acid sequence, byintroduction of nucleotide analogues (e.g. nucleotides with backbonemodifications, sugar modifications or base modifications) or byintroduction of stabilization sequences in the 3′- and/or5′-untranslated region of the inventive nucleic acid sequence.

As mentioned above, the inventive nucleic acid sequence as definedherein may contain nucleotide analogues/modifications e.g. backbonemodifications, sugar modifications or base modifications. A backbonemodification in connection with the present invention is a modificationin which phosphates of the backbone of the nucleotides contained ininventive nucleic acid sequence as defined herein are chemicallymodified. A sugar modification in connection with the present inventionis a chemical modification of the sugar of the nucleotides of theinventive nucleic acid sequence as defined herein. Furthermore, a basemodification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides of the nucleic acidmolecule of the inventive nucleic acid sequence. In this contextnucleotide analogues or modifications are preferably selected fromnucleotide analogues which are applicable for transcription and/ortranslation.

In a particular preferred embodiment of the first aspect of the presentinvention the herein defined nucleotide analogues/modifications areselected from base modifications which additionally increase theexpression of the encoded protein and which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-iodouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate,5-methyluridine-5′-triphosphate, 6-azacytidine-5′-triphosphate,6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

According to a further embodiment, the inventive nucleic acid sequenceas defined herein can contain a lipid modification. Such alipid-modified nucleic acid typically comprises a nucleic acid asdefined herein. Such a lipid-modified nucleic acid molecule of theinventive nucleic acid sequence as defined herein typically furthercomprises at least one linker covalently linked with that nucleic acidmolecule, and at least one lipid covalently linked with the respectivelinker. Alternatively, the lipid-modified nucleic acid moleculecomprises at least one nucleic acid molecule as defined herein and atleast one (bifunctional) lipid covalently linked (without a linker) withthat nucleic acid molecule. According to a third alternative, thelipid-modified nucleic acid molecule comprises a nucleic acid moleculeas defined herein, at least one linker covalently linked with thatnucleic acid molecule, and at least one lipid covalently linked with therespective linker, and also at least one (bifunctional) lipid covalentlylinked (without a linker) with that nucleic acid molecule. In thiscontext it is particularly preferred that the lipid modification ispresent at the terminal ends of a linear inventive nucleic acidsequence.

According to another preferred embodiment of the first aspect of theinvention, the inventive nucleic acid sequence as defined herein,particularly if provided as an (m)RNA, can therefore be stabilizedagainst degradation by RNases by the addition of a so-called “5′ CAP”structure.

According to a further preferred embodiment of the first aspect of theinvention, the inventive nucleic acid sequence as defined herein can bemodified by a sequence of at least 10 cytidines, preferably at least 20cytidines, more preferably at least 30 cytidines (so-called “poly(C)sequence”). Particularly, the inventive nucleic acid sequence maycontain or code for a poly(C) sequence of typically about 10 to 200cytidine nucleotides, preferably about 10 to 100 cytidine nucleotides,more preferably about 10 to 70 cytidine nucleotides or even morepreferably about 20 to 50 or even 20 to 30 cytidine nucleotides. Thispoly(C) sequence is preferably located 3′ of the coding region comprisedin the inventive nucleic acid according to the first aspect of thepresent invention.

In a particularly preferred embodiment of the present invention, the G/Ccontent of the coding region, encoding at least one peptide or proteinwhich comprises a therapeutic protein or a fragment, variant orderivative thereof of the inventive nucleic acid sequence as definedherein, is modified, particularly increased, compared to the G/C contentof its particular wild type coding region, i.e. the unmodified codingregion. The encoded amino acid sequence of the coding region ispreferably not modified compared to the coded amino acid sequence of theparticular wild type coding region.

The modification of the G/C-content of the coding region of theinventive nucleic acid sequence as defined herein is based on the factthat the sequence of any mRNA region to be translated is important forefficient translation of that mRNA. Thus, the composition and thesequence of various nucleotides are important. In particular, mRNAsequences having an increased G (guanosine)/C (cytosine) content aremore stable than mRNA sequences having an increased A (adenosine)/U(uracil) content. According to the invention, the codons of the codingregion are therefore varied compared to its wild type coding region,while retaining the translated amino acid sequence, such that theyinclude an increased amount of G/C nucleotides. In respect to the factthat several codons code for one and the same amino acid (so-calleddegeneration of the genetic code), the most favourable codons for thestability can be determined (so-called alternative codon usage).

Depending on the amino acid to be encoded by the coding region of theinventive nucleic acid sequence as defined herein, there are variouspossibilities for modification of the nucleic acid sequence, e.g. thecoding region, compared to its wild type coding region. In the case ofamino acids which are encoded by codons which contain exclusively G or Cnucleotides, no modification of the codon is necessary. Thus, the codonsfor Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC orGGG) require no modification, since no A or U is present.

In contrast, codons which contain A and/or U nucleotides can be modifiedby substitution of other codons which code for the same amino acids butcontain no A and/or U. Examples of these are:

-   the codons for Pro can be modified from CCU or CCA to CCC or CCG;-   the codons for Arg can be modified from CGU or CGA or AGA or AGG to    CGC or CGG;-   the codons for Ala can be modified from GCU or GCA to GCC or GCG;-   the codons for Gly can be modified from GGU or GGA to GGC or GGG.

In other cases, although A or U nucleotides cannot be eliminated fromthe codons, it is however possible to decrease the A and U content byusing codons which contain a lower content of A and/or U nucleotides.Examples of these are:

-   the codons for Phe can be modified from UUU to UUC;-   the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUC    or CUG;-   the codons for Ser can be modified from UCU or UCA or AGU to UCC,    UCG or AGC;-   the codon for Tyr can be modified from UAU to UAC;-   the codon for Cys can be modified from UGU to UGC;-   the codon for His can be modified from CAU to CAC;-   the codon for Gln can be modified from CAA to CAG;-   the codons for Ile can be modified from AUU or AUA to AUC;-   the codons for Thr can be modified from ACU or ACA to ACC or ACG;-   the codon for Asn can be modified from AAU to AAC;-   the codon for Lys can be modified from AAA to AAG;-   the codons for Val can be modified from GUU or GUA to GUC or GUG;-   the codon for Asp can be modified from GAU to GAC;-   the codon for Glu can be modified from GAA to GAG;-   the stop codon UAA can be modified to UAG or UGA.

In the case of the codons for Met (AUG) and Trp (UGG), on the otherhand, there is no possibility of sequence modification.

The substitutions listed above can be used either individually or in allpossible combinations to increase the G/C content of the coding regionof the inventive nucleic acid sequence as defined herein, compared toits particular wild type coding region (i.e. the original sequence).Thus, for example, all codons for Thr occurring in the wild typesequence can be modified to ACC (or ACG).

In the above context, codons present in mRNA are shown. Thereforeuridine present in an mRNA may also be present as thymidine in therespective DNA coding for the particular mRNA.

Preferably, the G/C content of the coding region of the inventivenucleic acid sequence as defined herein is increased by at least 7%,more preferably by at least 15%, particularly preferably by at least20%, compared to the G/C content of the wild type coding region.According to a specific embodiment at least 5%, 10%, 20%, 30%, 40%, 50%,60%, more preferably at least 70%, even more preferably at least 80% andmost preferably at least 90%, 95% or even 100% of the substitutablecodons in the coding region encoding at least one peptide or proteinwhich comprises a therapeutic protein or a fragment, variant orderivative thereof are substituted, thereby increasing the G/C contentof said coding region.

In this context, it is particularly preferable to increase the G/Ccontent of the coding region of the inventive nucleic acid sequence asdefined herein, to the maximum (i.e. 100% of the substitutable codons),compared to the wild type coding region.

According to the invention, a further preferred modification of thecoding region encoding at least one peptide or protein which comprises atherapeutic protein or a fragment, variant or derivative thereof of theinventive nucleic acid sequence as defined herein, is based on thefinding that the translation efficiency is also determined by adifferent frequency in the occurrence of tRNAs in cells. Thus, ifso-called “rare codons” are present in the coding region of theinventive nucleic acid sequence as defined herein, to an increasedextent, the corresponding modified nucleic acid sequence is translatedto a significantly poorer degree than in the case where codons codingfor relatively “frequent” tRNAs are present.

In this context the coding region of the inventive nucleic acid sequenceis preferably modified compared to the corresponding wild type codingregion such that at least one codon of the wild type sequence whichcodes for a tRNA which is relatively rare in the cell is exchanged for acodon which codes for a tRNA which is relatively frequent in the celland carries the same amino acid as the relatively rare tRNA. By thismodification, the coding region of the inventive nucleic acid sequenceas defined herein, is modified such that codons for which frequentlyoccurring tRNAs are available are inserted. In other words, according tothe invention, by this modification all codons of the wild type codingregion which code for a tRNA which is relatively rare in the cell can ineach case be exchanged for a codon which codes for a tRNA which isrelatively frequent in the cell and which, in each case, carries thesame amino acid as the relatively rare tRNA.

Which tRNAs occur relatively frequently in the cell and which, incontrast, occur relatively rarely is known to a person skilled in theart; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Thecodons which use for the particular amino acid the tRNA which occurs themost frequently, e.g. the Gly codon, which uses the tRNA which occursthe most frequently in the (human) cell, are particularly preferred.

According to the invention, it is particularly preferable to link thesequential G/C content which is increased, in particular maximized, inthe coding region of the inventive nucleic acid sequence as definedherein, with the “frequent” codons without modifying the amino acidsequence of the peptide or protein encoded by the coding region of thenucleic acid sequence. This preferred embodiment allows provision of aparticularly efficiently translated and stabilized (modified) inventivenucleic acid sequence as defined herein.

According to another preferred embodiment of the first aspect of theinvention, the inventive nucleic acid sequence as defined herein,preferably has additionally at least one 5′ and/or 3′ stabilizingsequence. These stabilizing sequences in the 5′ and/or 3′ untranslatedregions have the effect of increasing the half-life of the nucleic acid,particularly of the mRNA in the cytosol. These stabilizing sequences canhave 100% sequence identity to naturally occurring sequences which occurin viruses, bacteria and eukaryotes, but can also be partly orcompletely synthetic. The untranslated sequences (UTR) of the(alpha-)globin gene, e.g. from Homo sapiens or Xenopus laevis may bementioned as an example of stabilizing sequences which can be used inthe present invention for a stabilized nucleic acid. Another example ofa stabilizing sequence has the general formula(C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC (SEQ ID NO: 55), which is containedin the 3′-UTRs of the very stable RNAs which code for (alpha-)globin,type(I)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf.Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Suchstabilizing sequences can of course be used individually or incombination with one another and also in combination with otherstabilizing sequences known to a person skilled in the art. In thiscontext it is particularly preferred that the 3′ UTR sequence of thealpha globin gene is located 3′ of the coding region encoding at leastone peptide or protein which comprises a therapeutic protein or afragment, variant or derivative thereof comprised in the inventivenucleic acid sequence according to the first aspect of the presentinvention.

Substitutions, additions or eliminations of bases are preferably carriedout with the inventive nucleic acid sequence as defined herein, using aDNA matrix for preparation of the nucleic acid sequence by techniques ofthe well-known site directed mutagenesis or with an oligonucleotideligation strategy (see e.g. Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., ColdSpring Harbor, N.Y., 2001).

Any of the above modifications may be applied to the inventive nucleicacid sequence as defined herein and further to any nucleic acid as usedin the context of the present invention and may be, if suitable ornecessary, be combined with each other in any combination, provided,these combinations of modifications do not interfere with each other inthe respective nucleic acid. A person skilled in the art will be able totake his choice accordingly.

Nucleic acid sequences used according to the present invention asdefined herein may be prepared using any method known in the art,including synthetic methods such as e.g. solid phase synthesis, as wellas in vitro methods, such as in vitro transcription reactions or in vivoreactions, such as in vivo propagation of DNA plasmids in bacteria.

In such a process, for preparation of the inventive nucleic acidsequence as defined herein, especially if the nucleic acid is in theform of an mRNA, a corresponding DNA molecule may be transcribed invitro. This DNA matrix preferably comprises a suitable promoter, e.g. aT7 or SP6 promoter, for in vitro transcription, which is followed by thedesired nucleotide sequence for the nucleic acid molecule, e.g. mRNA, tobe prepared and a termination signal for in vitro transcription. The DNAmolecule, which forms the matrix of the at least one RNA of interest,may be prepared by fermentative proliferation and subsequent isolationas part of a plasmid which can be replicated in bacteria. Plasmids whichmay be mentioned as suitable for the present invention are e.g. theplasmids pT7Ts (GenBank accession number U26404; Lai et al., Development1995, 121: 2349 to 2360), pGEM® series, e.g. pGEM®-1 (GenBank accessionnumber X65300; from Promega) and pSP64 (GenBank accession numberX65327); cf. also Mezei and Storts, Purification of PCR Products, in:Griffin and Griffin (ed.), PCR Technology: Current Innovation, CRCPress, Boca Raton, Fla., 2001.

The inventive nucleic acid sequence as defined herein as well asproteins or peptides as encoded by this nucleic acid sequence maycomprise fragments or variants of those sequences. Such fragments orvariants may typically comprise a sequence having a sequence identitywith one of the above mentioned nucleic acids, or with one of theproteins or peptides or sequences, if encoded by the inventive nucleicacid sequence, of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferablyat least 70%, more preferably at least 80%, equally more preferably atleast 85%, even more preferably at least 90% and most preferably atleast 95% or even 97%, 98% or 99%, to the entire wild type sequence,either on nucleic acid level or on amino acid level.

“Fragments” of proteins or peptides in the context of the presentinvention (e.g. as encoded by the inventive nucleic acid sequence asdefined herein) may comprise a sequence of a protein or peptide asdefined herein, which is, with regard to its amino acid sequence (or itsencoded nucleic acid molecule), N-terminally, C-terminally and/orintrasequentially truncated/shortened compared to the amino acidsequence of the original (native) protein (or its encoded nucleic acidmolecule). Such truncation may thus occur either on the amino acid levelor correspondingly on the nucleic acid level. A sequence identity withrespect to such a fragment as defined herein may therefore preferablyrefer to the entire protein or peptide as defined herein or to theentire (coding) nucleic acid molecule of such a protein or peptide.Likewise, “fragments” of nucleic acids in the context of the presentinvention may comprise a sequence of a nucleic acid as defined herein,which is, with regard to its nucleic acid molecule 5′-, 3′- and/orintrasequentially truncated/shortened compared to the nucleic acidmolecule of the original (native) nucleic acid molecule. A sequenceidentity with respect to a fragment as defined herein may thereforepreferably refer to the entire nucleic acid as defined herein and thepreferred sequence identity level typically is as indicated herein.Fragments have the same biological function or specific activity or atleast retain an activity of the natural full length protein of at least50%, more preferably at least 70%, even more preferably at least 90%(measured in an appropriate functional assay, e.g. an assay assessingthe enzymatic activity of the fragment of the therapeutic protein or thebinding activity thereof) as compared to the full-length native wtpeptide or protein, e.g. its specific antigenic or therapeutic property.Accordingly, in a preferred embodiment, the “fragment” is a portion ofthe full-length (naturally occurring) wt therapeutic protein, whichexerts therapeutic properties as indicated herein.

Furthermore also domains of a protein, like the extracellular domain,the intracellular domain or the transmembran domain and shortened ortruncated versions of a protein may be understood to comprise/correspondto a fragment of a protein.

“Variants” of proteins or peptides as defined in the context of thepresent invention may be encoded by the inventive nucleic acid sequenceas defined herein. Thereby, a protein or peptide may be generated,having an amino acid sequence which differs from the original sequencein one or more (2, 3, 4, 5, 6, 7 or more) mutation(s), such as one ormore substituted, inserted and/or deleted amino acid(s). The preferredlevel of sequence identity of “variants” in view of the full-lengthnatural wt protein sequence typically is as indicated herein.Preferably, variants have the same biological function or specificactivity or at least retain an activity of the natural full lengthprotein of at least 50%, more preferably at least 70%, even morepreferably at least 90% (measured in an appropriate functional assay,e.g. an assay assessing the enzymatic activity of the variant of thetherapeutic protein or the binding activity thereof) as compared to thefull-length native peptide or protein, e.g. its specific therapeuticproperty. Accordingly, in a preferred embodiment, the “variant” is avariant of a therapeutic protein, which exerts therapeutic properties tothe extent as indicated herein.

“Variants” of proteins or peptides as defined in the context of thepresent invention (e.g. as encoded by a nucleic acid as defined herein)may comprise conservative amino acid substitution(s) compared to theirnative, i.e. non-mutated physiological, sequence. Those amino acidsequences as well as their encoding nucleotide sequences in particularfall under the term variants as defined herein. Substitutions in whichamino acids, which originate from the same class, are exchanged for oneanother are called conservative substitutions. In particular, these areamino acids having aliphatic side chains, positively or negativelycharged side chains, aromatic groups in the side chains or amino acids,the side chains of which can enter into hydrogen bridges, e.g. sidechains which have a hydroxyl function. This means that e.g. an aminoacid having a polar side chain is replaced by another amino acid havinga likewise polar side chain, or, for example, an amino acidcharacterized by a hydrophobic side chain is substituted by anotheramino acid having a likewise hydrophobic side chain (e.g. serine(threonine) by threonine (serine) or leucine (isoleucine) by isoleucine(leucine)). Insertions and substitutions are possible, in particular, atthose sequence positions which cause no modification to thethree-dimensional structure or do not affect the binding region.Modifications to a three-dimensional structure by insertion(s) ordeletion(s) can easily be determined e.g. using CD spectra (circulardichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORDof Polypeptides, in: Modern Physical Methods in Biochemistry, Neubergeret al. (ed.), Elsevier, Amsterdam).

Furthermore, variants of proteins or peptides as defined herein, whichmay be encoded by the inventive nucleic acid sequence as defined herein,may also comprise those sequences, wherein nucleotides of the nucleicacid are exchanged according to the degeneration of the genetic code,without leading to an alteration of the respective amino acid sequenceof the protein or peptide, i.e. the amino acid sequence or at least partthereof may not differ from the original sequence in one or moremutation(s) within the above meaning.

In order to determine the percentage to which two sequences areidentical, e.g. nucleic acid sequences or amino acid sequences asdefined herein, preferably the amino acid sequences encoded by theinventive nucleic acid sequence as defined herein or the amino acidsequences themselves, the sequences can be aligned in order to besubsequently compared to one another. Therefore, e.g. a position of afirst sequence may be compared with the corresponding position of thesecond sequence. If a position in the first sequence is occupied by thesame component as is the case at a position in the second sequence, thetwo sequences are identical at this position. If this is not the case,the sequences differ at this position. If insertions occur in the secondsequence in comparison to the first sequence, gaps can be inserted intothe first sequence to allow a further alignment. If deletions occur inthe second sequence in comparison to the first sequence, gaps can beinserted into the second sequence to allow a further alignment. Thepercentage to which two sequences are identical is then a function ofthe number of identical positions divided by the total number ofpositions including those positions which are only occupied in onesequence. The percentage to which two sequences are identical can bedetermined using a mathematical algorithm. A preferred, but notlimiting, example of a mathematical algorithm which can be used is thealgorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul etal. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm isintegrated in the BLAST program. Sequences which are identical to thesequences of the present invention to a certain extent can be identifiedby this program.

The inventive nucleic acid sequence as defined herein may encodederivatives of a peptide or protein. Such a derivative of a peptide orprotein is a molecule that is derived from another molecule, such assaid peptide or protein. A “derivative” typically contains thefull-length sequence of the natural wt peptide or protein and additionalsequence features, e.g. at the N- or at the C-terminus, which mayexhibit an additional function to the natural full-lengthpeptide/protein. Again such derivatives have the same biologicalfunction or specific activity or at least retain an activity of thenatural wt full-length protein of at least 50%, more preferably at least70%, even more preferably at least 90% (measured in an appropriatefunctional assay, see above, e.g. a binding or an enzyme activity assay)as compared to the full-length native peptide or protein, e.g. itsspecific therapeutic property. Thereby, a “derivative” of a peptide orprotein also encompasses (chimeric) fusion peptides/proteins comprisinga peptide or protein used in the present invention or a naturalfull-length protein (or variant/fragment thereof) fused to a distinctpeptide/protein awarding e.g. two or more biological functions to thefusion peptide/protein. For example, the fusion may comprise a label,such as, for example, an epitope, e.g., a FLAG epitope or a V5 epitopeor an HA epitope. For example, the epitope is a FLAG epitope. Such a tagis useful for, for example, purifying the fusion protein.

In this context, a “variant” of a protein or peptide may have at least70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over astretch of 10, 20, 30, 50, 75 or 100 amino acids of such protein orpeptide. Analogously, a “variant” of a nucleic acid sequence, orparticularly, a fragment, may have at least 70%, 75%, 80%, 85%, 90%,95%, 98% or 99% nucleotide identity over a stretch of 10, 20, 30, 50, 75or 100 nucleotide of such nucleic acid sequence; typically, however,referring to the naturally occurring full-length sequences. In case of“fragments” typically, sequence identity is determined for the fragmentover the length (of the fragment) on the portion of the full-lengthprotein (reflecting the same length as the fragment), which exhibits thehighest level of sequence identity.

In a further preferred embodiment of the first aspect of the presentinvention the inventive nucleic acid sequence is associated with avehicle, transfection or complexation agent for increasing thetransfection efficiency and/or the immunostimulatory properties of theinventive nucleic acid sequence. Particularly preferred agents in thiscontext suitable for increasing the transfection efficiency are cationicor polycationic compounds, including protamine, nucleoline, spermine orspermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSVVP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs),PpT620, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s),Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, pIs1, FGF, Lactoferrin, Transportan, Buforin-2,Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, or histones.Additionally, preferred cationic or polycationic proteins or peptidesmay be selected from the following proteins or peptides having thefollowing total formula:(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), whereinl+m+n+o+x=8-15, and l, m, n or o independently of each other may be anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3 or 4, provided, that the overall contentof Xaa does not exceed 50% of all amino acids of the oligopeptide.Particularly preferred cationic peptides in this context are e.g. Arg₇,Arg₈, Arg₉, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc. Furtherpreferred cationic or polycationic compounds, which can be used astransfection agent may include cationic polysaccharides, for examplechitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI),cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g polyethyleneglycole); etc.

In this context it is particularly preferred that the inventive nucleicacid is complexed at least partially with a cationic or polycationiccompound, preferably cationic proteins or peptides. Partially means thatonly a part of the inventive nucleic acid is complexed with a cationicor polycationic compound and that the rest of the inventive nucleic acidis in uncomplexed form (“free”). Preferably the ratio of complexednucleic acid to: free nucleic acid is selected from a range. of about5:1 (w/w) to about 1:10 (w/w), more preferably from a range of about 4:1(w/w) to about 1:8 (w/w), even more preferably from a range of about 3:1(w/w) to about 1:5 (w/w) or 1:3 (w/w), and most preferably the ratio ofcomplexed nucleic acid to free nucleic acid is selected from a ratio ofabout 1:1 (w/w).

It is preferred that the nucleic acid sequence of the invention isprovided in either naked form or complexed, e.g. by polycationiccompounds of whatever chemical structure, preferably polycationic(poly)peptides or synthetic polycationic compounds. Preferably, thenucleic acid sequence is not provided together with a packaging cell.

In a further aspect the invention provides for a composition or kit orkit of parts comprising a plurality or more than one, preferably 2 to10, more preferably 2 to 5, most preferably 2 to 4 of the inventivenucleic acid sequences as defined herein. These inventive compositionscomprise more than one inventive nucleic acid sequences, preferablyencoding different peptides or proteins which comprise preferablydifferent therapeutic proteins or fragments, variants or derivativesthereof.

According to a further aspect, the present invention also provides amethod for increasing the expression of an encoded peptide or proteincomprising the steps, e.g. a) providing the inventive nucleic acidsequence as defined herein or the inventive composition comprising aplurality (which means typically more than 1, 2, 3, 4, 5, 6 or more than10 nucleic acids, e.g. 2 to 10, preferably 2 to 5 nucleic acids) ofinventive nucleic acid sequences as defined herein, b) applying oradministering the inventive nucleic acid sequence as defined herein orthe inventive composition comprising a plurality of inventive nucleicacid sequences as defined herein to an expression system, e.g. to acell-free expression system, a cell (e.g. an expression host cell or asomatic cell), a tissue or an organism. The method may be applied forlaboratory, for research, for diagnostic, for commercial production ofpeptides or proteins and/or for therapeutic purposes. In this context,typically after preparing the inventive nucleic acid sequence as definedherein or of the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein, it is typicallyapplied or administered to a cell-free expression system, a cell (e.g.an expression host cell or a somatic cell), a tissue or an organism,e.g. in naked or complexed form or as a pharmaceutical composition asdescribed herein, preferably via transfection or by using any of theadministration modes as described herein. The method may be carried outin vitro, in vivo or ex vivo. The method may furthermore be carried outin the context of the treatment of a specific disease, preferably asdefined herein.

In this context in vitro is defined herein as transfection ortransduction of the inventive nucleic acid as defined herein or of theinventive composition comprising a plurality of inventive nucleic acidsequences as defined herein into cells in culture outside of anorganism; in vivo is defined herein as transfection or transduction ofthe inventive nucleic acid or of the inventive composition comprising aplurality of inventive nucleic acid sequences (which means typicallymore than 1, 2, 3, 4, 5, 6 or more than 10 nucleic acids, e.g. 2 to 10,preferably 2 to 5 nucleic acids) into cells by application of theinventive nucleic acid or of the inventive composition to the wholeorganism or individual and ex vivo is defined herein as transfection ortransduction of the inventive nucleic acid or of the inventivecomposition comprising a plurality of inventive nucleic acid sequencesinto cells outside of an organism or individual and subsequentapplication of the transfected cells to the organism or individual.

Likewise, according to another aspect, the present invention alsoprovides the use of the inventive nucleic acid sequence as definedherein or of the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein, preferably fordiagnostic or therapeutic purposes, for increasing the expression of anencoded peptide or protein, particularly in gene therapy e.g. byapplying or administering the inventive nucleic acid sequence as definedherein or of the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein, e.g. to a cell-freeexpression system, a cell (e.g. an expression host cell or a somaticcell), a tissue or an organism. The use may be applied for laboratory,for research, for diagnostic for commercial production of peptides orproteins and/or for therapeutic purposes, preferably for gene therapy.In this context, typically after preparing the inventive nucleic acidsequence as defined herein or of the inventive composition comprising aplurality of inventive nucleic acid sequences as defined herein, it istypically applied or administered to a cell-free expression system, acell (e.g. an expression host cell or a somatic cell), a tissue or anorganism, preferably in naked form or complexed form, or as apharmaceutical composition as described herein, preferably viatransfection or by using any of the administration modes as describedherein. The use may be carried out in vitro, in vivo or ex vivo. The usemay furthermore be carried out in the context of the treatment of aspecific disease, preferably as defined herein.

In yet another aspect the present invention also relates to an inventiveexpression system comprising an inventive nucleic acid sequence orexpression vector or plasmid according to the first aspect of thepresent invention. In this context the expression system may be acell-free expression system (e.g. an in vitro transcription/translationsystem), a cellular expression system (e.g. mammalian cells like CHOcells, insect cells, yeast cells, bacterial cells like E. coli) ororganisms used for expression of peptides or proteins (e.g. plants oranimals like cows).

Additionally, according to another aspect, the present invention alsorelates to the use of the inventive nucleic acid as defined herein or ofthe inventive composition comprising a plurality of inventive nucleicacid sequences as defined herein for the preparation of a pharmaceuticalcomposition for increasing the expression of an encoded peptide orprotein, particularly for use in gene therapy, e.g. for treating adisease, preferably as defined herein, e.g. applying or administeringthe inventive nucleic acid as defined herein or of the inventivecomposition comprising a plurality of inventive nucleic acid sequencesas defined herein to a cell (e.g. an expression host cell or a somaticcell), a tissue or an organism, preferably in naked form or complexedform or as a pharmaceutical composition as described herein, morepreferably using any of the administration modes as described herein.

Accordingly, in a particular preferred aspect, the present inventionalso provides a pharmaceutical composition, comprising an inventivenucleic acid as defined herein or an inventive composition comprising aplurality of inventive nucleic acid sequences as defined herein andoptionally a pharmaceutically acceptable carrier and/or vehicle.

As a first ingredient, the inventive pharmaceutical compositioncomprises at least one inventive nucleic acid as defined herein.

As a second ingredient the inventive pharmaceutical composition mayoptional comprise at least one additional pharmaceutically activecomponent. A pharmaceutically active component in this connection is acompound that has a therapeutic effect to heal, ameliorate or prevent aparticular indication or disease as mentioned herein. Such compoundsinclude, without implying any limitation, peptides or proteins,preferably as defined herein, nucleic acids, preferably as definedherein, (therapeutically active) low molecular weight organic orinorganic compounds (molecular weight less than 5000, preferably lessthan 1000), sugars, antigens or antibodies, preferably as definedherein, therapeutic agents already known in the prior art, antigeniccells, antigenic cellular fragments, cellular fractions; cell wallcomponents (e.g. polysaccharides), modified, attenuated or de-activated(e.g. chemically or by irradiation) pathogens (virus, bacteria etc.),adjuvants, preferably as defined herein, etc.

Furthermore, the inventive pharmaceutical composition may comprise apharmaceutically acceptable carrier and/or vehicle. In the context ofthe present invention, a pharmaceutically acceptable carrier typicallyincludes the liquid or non-liquid basis of the inventive pharmaceuticalcomposition. If the inventive pharmaceutical composition is provided inliquid form, the carrier will typically be pyrogen-free water; isotonicsaline or buffered (aqueous) solutions, e.g phosphate, citrate etc.buffered solutions. The injection buffer may be hypertonic, isotonic orhypotonic with reference to the specific reference medium, i.e. thebuffer may have a higher, identical or lower salt content with referenceto the specific reference medium, wherein preferably such concentrationsof the afore mentioned salts may be used, which do not lead to damage ofcells due to osmosis or other concentration effects. Reference media aree.g. liquids occurring in “in vivo” methods, such as blood, lymph,cytosolic liquids, or other body liquids, or e.g. liquids, which may beused as reference media in “in vitro” methods, such as common buffers orliquids. Such common buffers or liquids are known to a skilled person.Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well for the inventivepharmaceutical composition, which are suitable for administration to apatient to be treated. The term “compatible” as used here means thatthese constituents of the inventive pharmaceutical composition arecapable of being mixed with the inventive nucleic acid as defined hereinin such a manner that no interaction occurs which would substantiallyreduce the pharmaceutical effectiveness of the inventive pharmaceuticalcomposition under typical use conditions.

According to a specific embodiment, the inventive pharmaceuticalcomposition may comprise an adjuvant. In this context, an adjuvant maybe understood as any compound, which is suitable to initiate or increasean immune response of the innate immune system, i.e. a non-specificimmune response. With other words, when administered, the inventivepharmaceutical composition preferably elicits an innate immune responsedue to the adjuvant, optionally contained therein. Preferably, such anadjuvant may be selected from an adjuvant known to a skilled person andsuitable for the present case, i.e. supporting the induction of aninnate immune response in a mammal, e.g. an adjuvant protein as definedabove or an adjuvant as defined in the following.

Particularly preferred as adjuvants suitable for depot and delivery arecationic or polycationic compounds as defined above for the inventivenucleic acid sequence as vehicle, transfection or compl exati on agent.

Further additives which may be included in the inventive pharmaceuticalcomposition are emulsifiers, such as, for example, Tween®; wettingagents, such as, for example, sodium lauryl sulfate; colouring agents;taste-imparting agents, pharmaceutical carriers; tablet-forming agents;stabilizers; antioxidants; preservatives.

The inventive pharmaceutical composition can also additionally containany further compound, which is known to be immunostimulating due to itsbinding affinity (as ligands) to human Toll-like receptors TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

The inventive pharmaceutical composition may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term parenteralas used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional, intracranial, transdermal, intradermal,intrapulmonal, intraperitoneal, intracardial, intraarterial, andsublingual injection or infusion techniques.

Preferably, the inventive pharmaceutical composition may be administeredby parenteral injection, more preferably by subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, intracranial, transdermal,intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, and sublingual injection or via infusion techniques.Particularly preferred is intradermal and intramuscular injection.Sterile injectable forms of the inventive pharmaceutical compositionsmay be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents.

The inventive pharmaceutical composition as defined herein may also beadministered orally in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions.

The inventive pharmaceutical composition may also be administeredtopically, especially when the target of treatment includes areas ororgans readily accessible by topical application, e.g. includingdiseases of the skin or of any other accessible epithelial tissue.Suitable topical formulations are readily prepared for each of theseareas or organs. For topical applications, the inventive pharmaceuticalcomposition may be formulated in a suitable ointment, containing theinventive nucleic acid as defined herein suspended or dissolved in oneor more carriers.

The inventive pharmaceutical composition typically comprises a “safe andeffective amount” of the components of the inventive pharmaceuticalcomposition, particularly of the inventive nucleic acid sequence(s) asdefined herein. As used herein, a “safe and effective amount” means anamount of the inventive nucleic acid sequence(s) as defined herein assuch that is sufficient to significantly induce a positive modificationof a disease or disorder as defined herein. At the same time, however, a“safe and effective amount” is small enough to avoid seriousside-effects and to permit a sensible relationship between advantage andrisk. The determination of these limits typically lies within the scopeof sensible medical judgment.

The present invention furthermore provides several applications and usesof the inventive nucleic acid sequence as defined herein, of theinventive composition comprising a plurality of inventive nucleic acidsequences as defined herein, of the inventive pharmaceuticalcomposition, comprising the inventive nucleic acid sequence as definedherein or of kits comprising same.

According to one specific aspect, the present invention is directed tothe first medical use of the inventive nucleic acid sequence as definedherein or of the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein as a medicament,particularly in gene therapy, preferably for the treatment of diseasesas defined herein.

According to another aspect, the present invention is directed to thesecond medical use of the inventive nucleic acid sequence as definedherein or of the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein, for the treatment ofdiseases as defined herein, preferably to the use of the inventivenucleic acid sequence as defined herein, of the inventive compositioncomprising a plurality of inventive nucleic acid sequences as definedherein, of a pharmaceutical composition comprising same or of kitscomprising same for the preparation of a medicament for the prophylaxis,treatment and/or amelioration of diseases as defined herein. Preferably,the pharmaceutical composition is used or to be administered to apatient in need thereof for this purpose.

Preferably, diseases as mentioned herein are preferably selected frominfectious diseases, neoplasms (e.g. cancer or tumour diseases),diseases of the blood and blood-forming organs, endocrine, nutritionaland metabolic diseases, diseases of the nervous system, diseases of thecirculatory system, diseases of the respiratory system, diseases of thedigestive system, diseases of the skin and subcutaneous tissue, diseasesof the musculoskeletal system and connective tissue, and diseases of thegenitourinary system.

In this context particularly preferred are inherited diseases selectedfrom: 1p36 deletion syndrome; 18p deletion syndrome; 21-hydroxylasedeficiency; 45,X (Turner syndrome); 47,XX,+21 (Down syndrome); 47,XXX(triple X syndrome); 47,XXY (Klinefelter syndrome); 47,XY,+21 (Downsyndrome); 47,XYY syndrome; 5-ALA dehydratase-deficient porphyria (ALAdehydratase deficiency); 5-aminolaevulinic dehydratase deficiencyporphyria (ALA dehydratase deficiency); 5p deletion syndrome (Cri duchat) 5p-syndrome (Cri du chat); A-T (ataxia-telangiectasia); AAT(alpha-1 antitrypsin deficiency); Absence of vas deferens (congenitalbilateral absence of vas deferens); Absent vasa (congenital bilateralabsence of vas deferens); aceruloplasminemia; ACG2 (achondrogenesis typeII); ACH (achondroplasia); Achondrogenesis type II; achondroplasia; Acidbeta-glucosidase deficiency (Gaucher disease type 1);Acrocephalosyndactyly (Apert) (Apert syndrome); acrocephalosyndactyly,type V (Pfeiffer syndrome); Acrocephaly (Apert syndrome); Acute cerebralGaucher's disease (Gaucher disease type 2); acute intermittentporphyria; ACY2 deficiency (Canavan disease); AD (Alzheimer's disease);Adelaide-type craniosynostosis (Muenke syndrome); Adenomatous PolyposisColi (familial adenomatous polyposis); Adenomatous Polyposis of theColon (familial adenomatous polyposis); ADP (ALA dehydratasedeficiency); adenylosuccinate lyase deficiency; Adrenal gland disorders(21-hydroxylase deficiency); Adrenogenital syndrome (21-hydroxylasedeficiency); Adrenoleukodystrophy; AIP (acute intermittent porphyria);AIS (androgen insensitivity syndrome); AKU (alkaptonuria); ALAdehydratase porphyria (ALA dehydratase deficiency); ALA-D porphyria (ALAdehydratase deficiency); ALA dehydratase deficiency; Alcaptonuria(alkaptonuria); Alexander disease; alkaptonuria; Alkaptonuric ochronosis(alkaptonuria); alpha-1 antitrypsin deficiency; alpha-1 proteinaseinhibitor (alpha-1 antitrypsin deficiency); alpha-1 related emphysema(alpha-1 antitrypsin deficiency); Alpha-galactosidase A deficiency(Fabry disease); ALS (amyotrophic lateral sclerosis); Alstrom syndrome;ALX (Alexander disease); Alzheimer disease; Amelogenesis Imperfecta;Amino levulinic acid dehydratase deficiency (ALA dehydratasedeficiency); Aminoacylase 2 deficiency (Canavan disease); amyotrophiclateral sclerosis; Anderson-Fabry disease (Fabry disease); androgeninsensitivity syndrome; Anemia; Anemia, hereditary sideroblastic(X-linked sideroblastic anemia); Anemia, sex-linked hypochromicsideroblastic (X-linked sideroblastic anemia); Anemia, splenic, familial(Gaucher disease); Angelman syndrome; Angiokeratoma Corporis Diffusum(Fabry's disease); Angiokeratoma diffuse (Fabry's disease); Angiomatosisretinae (von Hippel-Lindau disease); ANH1 (X-linked sideroblasticanemia); APC resistance, Leiden type (factor V Leiden thrombophilia);Apert syndrome; AR deficiency (androgen insensitivity syndrome); AR-CMT2ee (Charcot-Mare-Tooth disease, type 2); Arachnodactyly (Marfansyndrome); ARNSHL (Nonsyndromic deafness#autosomal recessive);Arthro-ophthalmopathy, hereditary progressive (Sticklersyndrome#COL2A1); Arthrochalasis multiplex congenita (Ehlers-Danlossyndrome#arthrochalasia type); AS (Angelman syndrome); Asp deficiency(Canavan disease); Aspa deficiency (Canavan disease); Aspartoacylasedeficiency (Canavan disease); ataxia-telangiectasia;Autism-Dementia-Ataxia-Loss of Purposeful Hand Use syndrome (Rettsyndrome); autosomal dominant juvenile ALS (amyotrophic lateralsclerosis, type 4); Autosomal dominant opitz G/BBB syndrome (22q11.2deletion syndrome); autosomal recessive form of juvenile ALS type 3(Amyotrophic lateral sclerosis#type 2); Autosomal recessive nonsyndromichearing loss (Nonsyndromic deafness#autosomal recessive); AutosomalRecessive Sensorineural Hearing Impairment and Goiter (Pendredsyndrome); AxD (Alexander disease); Ayerza syndrome (primary pulmonaryhypertension); B variant of the Hexosaminidase GM2 gangliosidosis(Sandhoff disease); BANF (neurofibromatosis 2); Beare-Stevenson cutisgyrata syndrome; Benign paroxysmal peritonitis (Mediterranean fever,familial); Benjamin syndrome; beta thalassemia; BH4 Deficiency(tetrahydrobiopterin deficiency); Bilateral Acoustic Neurofibromatosis(neurofibromatosis 2); biotinidase deficiency; bladder cancer; Bleedingdisorders (factor V Leiden thrombophilia); Bloch-Sulzberger syndrome(incontinentia pigmenti); Bloom syndrome; Bone diseases; Bone marrowdiseases (X-linked sideroblastic anemia); Bonnevie-Ullrich syndrome(Turner syndrome); Bourneville disease (tuberous sclerosis); Bournevillephakomatosis (tuberous sclerosis); Brain diseases (prion disease);breast cancer; Birt-Hogg-Dubé syndrome; Brittle bone disease(osteogenesis imperfecta); Broad Thumb-Hallux syndrome (Rubinstein-Taybisyndrome); Bronze Diabetes (hemochromatosis); Bronzed cirrhosis(hemochromatosis); Bulbospinal muscular atrophy, X-linked (Kennedydisease); Burger-Grutz syndrome (lipoprotein lipase deficiency,familial); CADASIL; CGD Chronic Granulomatous Disorder; Camptomelicdysplasia; Canavan disease; Cancer; Cancer Family syndrome (hereditarynonpolyposis colorectal cancer); Cancer of breast (breast cancer);Cancer of the bladder (bladder cancer); Carboxylase Deficiency,Multiple, Late-Onset (biotinidase deficiency); Cardiomyopathy (Noonansyndrome); Cat cry syndrome (Cri du chat); CAVD (congenital bilateralabsence of vas deferens); Caylor cardiofacial syndrome (22q11.2 deletionsyndrome); CBAVD (congenital bilateral absence of vas deferens); CeliacDisease; CEP (congenital erythropoietic porphyria); Ceramidetrihexosidase deficiency (Fabry disease); Cerebelloretinal Angiomatosis,familial (von Hippel-Lindau disease); Cerebral arteriopathy withsubcortical infarcts and leukoencephalopathy (CADASIL); Cerebralautosomal dominant ateriopathy with subcortical infarcts andleukoencephalopathy (CADASIL); Cerebral sclerosis (tuberous sclerosis);Cerebroatrophic Hyperammonemia (Rett syndrome); Cerebroside Lipidosissyndrome (Gaucher disease); CF (cystic fibrosis); CH (congenitalhypothyroidism); Charcot disease (amyotrophic lateral sclerosis);Charcot-Marie-Tooth disease; Chondrodystrophia (achondroplasia);Chondrodystrophy syndrome (achondroplasia); Chondrodystrophy withsensorineural deafness (otospondylomegaepiphyseal dysplasia);Chondrogenesis imperfecta (achondrogenesis, type II); Choreoathetosisself-mutilation hyperuricemia syndrome (Lesch-Nyhan syndrome); ClassicGalactosemia (galactosemia); Classical Ehlers-Danlos syndrome(Ehlers-Danlos syndrome#classical type); Classical Phenylketonuria(phenylketonuria); Cleft lip and palate (Stickler syndrome); Cloverleafskull with thanatophoric dwarfism (Thanatophoric dysplasia#type 2); CLS(Coffin-Lowry syndrome); CMT (Charcot-Marie-Tooth disease); Cockaynesyndrome; Coffin-Lowry syndrome; collagenopathy, types II and XI; ColonCancer, familial Nonpolyposis (hereditary nonpolyposis colorectalcancer); Colon cancer, familial (familial adenomatous polyposis);Colorectal Cancer; Complete HPRT deficiency (Lesch-Nyhan syndrome);Complete hypoxanthine-guanine phosphoribosy transferase deficiency(Lesch-Nyhan syndrome); Compression neuropathy (hereditary neuropathywith liability to pressure palsies); Congenital adrenal hyperplasia(21-hydroxylase deficiency); congenital bilateral absence of vasdeferens (Congenital absence of the vas deferens); Congenitalerythropoietic porphyria; Congenital heart disease; Congenitalhypomyelination (Charcot-Marie-Tooth disease#Type 1/Charcot-Marie-Toothdisease#Type 4); Congenital hypothyroidism; Congenital methemoglobinemia(Methemoglobinemia#Congenital methaemoglobinaemia); Congenitalosteosclerosis (achondroplasia); Congenital sideroblastic anaemia(X-linked sideroblastic anemia); Connective tissue disease; Conotruncalanomaly face syndrome (22q11.2 deletion syndrome); Cooley's Anemia (betathalassemia); Copper storage disease (Wilson disease); Copper transportdisease (Menkes disease); Coproporphyria, hereditary (hereditarycoproporphyria); Coproporphyrinogen oxidase deficiency (hereditarycoproporphyria); Cowden syndrome; CPO deficiency (hereditarycoproporphyria); CPRO deficiency (hereditary coproporphyria); CPXdeficiency (hereditary coproporphyria); Craniofacial dysarthrosis(Crouzon syndrome); Craniofacial Dysostosis (Crouzon syndrome);Cretinism (congenital hypothyroidism); Creutzfeldt-Jakob disease (priondisease); Cri du chat (Crohn's disease, fibrostenosing); Crouzonsyndrome; Crouzon syndrome with acanthosis nigricans(Crouzonodermoskeletal syndrome); Crouzonodermoskeletal syndrome; CS(Cockayne syndrome)(Cowden syndrome); Curschmann-Batten-Steinertsyndrome (myotonic dystrophy); cutis gyrata syndrome of Beare-Stevenson(Beare-Stevenson cutis gyrata syndrome); Disorder Mutation Chromosome;D-glycerate dehydrogenase deficiency (hyperoxaluria, primary); Dappledmetaphysis syndrome (spondyloepimetaphyseal dysplasia, Strudwick type);DAT—Dementia Alzheimer's type (Alzheimer disease); Genetichypercalciuria (Dent's disease); DBMD (muscular dystrophy, Duchenne andBecker types); Deafness with goiter (Pendred syndrome);Deafness-retinitis pigmentosa syndrome (Usher syndrome); Deficiencydisease, Phenylalanine Hydroxylase (phenylketonuria); Degenerative nervediseases; de Grouchy syndrome 1 (De Grouchy Syndrome); Dejerine-Sottassyndrome (Charcot-Marie-Tooth disease); Delta-aminolevulinatedehydratase deficiency porphyria (ALA dehydratase deficiency); Dementia(CADASIL); demyelinogenic leukodystrophy (Alexander di sea se);Dermatosparactic type of Ehlers-Danlos syndrome (Ehlers-Danlossyndrome#dermatosparaxi s type); Dermatosparaxi s (Ehlers-Danlossyndrome#dermatosparaxi s type) ; developmental disabilities; dHMN(Amyotrophic lateral sclerosis#type 4); DHMN-V (distal spinal muscularatrophy, type V); DHTR deficiency (androgen insensitivity syndrome);Diffuse Globoid Body Sclerosis (Krabbe disease); DiGeorge syndrome;Dihydrotestosterone receptor deficiency (androgen insensitivitysyndrome); distal spinal muscular atrophy, type V; DM1 (Myotonicdystrophy#typel); DM2 (Myotonic dystrophy#type2); Down syndrome; DSMAV(distal spinal muscular atrophy, type V); DSN (Charcot-Marie-Toothdisease#type 4); DSS (Charcot-Marie-Tooth disease, type 4);Duchenne/Becker muscular dystrophy (muscular dystrophy, Duchenne andBecker types); Dwarf, achondroplastic (achondroplasia); Dwarf,thanatophoric (thanatophoric dysplasia); Dwarfism; Dwarfism-retinalatrophy-deafness syndrome (Cockayne syndrome); dysmyelinogenicleukodystrophy (Alexander disease); Dystrophia myotonica (myotonicdystrophy); dystrophia retinae pigmentosa-dysostosis syndrome (Ushersyndrome); Early-Onset familial alzheimer disease (EOFAD) (Alzheimerdisease); EDS (Ehlers-Danlos syndrome); Ehlers-Danlos syndrome;Ekman-Lob stein disease (osteogenesi s imperfecta); Entrapmentneuropathy (hereditary neuropathy with liability to pressure palsies);Epiloia (tuberous sclerosis); EPP (erythropoietic protoporphyria);Erythroblastic anemia (beta thal as semi a); Erythrohepaticprotoporphyria (erythropoietic protoporphyria); Erythroid5-aminolevulinate synthetase deficiency (X-linked sideroblastic anemia);Erythropoietic porphyria (congenital erythropoietic p orphyri a);Erythropoietic protoporphyria; Erythropoietic uroporphyria (congenitalerythropoietic porphyria); Eye cancer (retinoblastoma FA—Friedreichataxia); Fabry disease; Facial injuries and disorders; Factor V Leidenthrombophilia; FAL S (amyotrophic lateral sclerosis); familial acousticneuroma (neurofibromatosis type II); familial adenomatous polyposis;familial Alzheimer disease (FAD) (Alzheimer disease); familialamyotrophic lateral sclerosis (amyotrophic lateral sclerosis); familialdysautonomia; familial fat-induced hypertriglyceridemia (lipoproteinlipase deficiency, familial); familial hemochromatosis(hemochromatosis); familial LPL deficiency (lipoprotein lipasedeficiency, familial); familial nonpolyposis colon cancer (hereditarynonpolyposis colorectal cancer); familial paroxysmal polyserositis(Mediterranean fever, familial); familial PCT (porphyria cutanea tarda);familial pressure sensitive neuropathy (hereditary neuropathy withliability to pressure palsies); familial primary pulmonary hypertension(FPPH) (primary pulmonary hypertension); Familial Turner syndrome(Noonan syndrome); familial vascular leukoencephalopathy (CADASIL); FAP(familial adenomatous polyposis); FD (familial dysautonomia); Femalepseudo-Turner syndrome (Noonan syndrome); Ferrochelatase deficiency(erythropoietic protoporphyria); ferroportin disease(Haemochromatosis#type 4); Fever (Mediterranean fever, familial); FGsyndrome; FGFR3-associated coronal synostosis (Muenke syndrome);Fibrinoid degeneration of astrocytes (Alexander disease); Fibrocysticdisease of the pancreas (cystic fibrosis); FMF (Mediterranean fever,familial); Folling disease (phenylketonuria); fra(X) syndrome (fragile Xsyndrome); fragile X syndrome; Fragilitas ossium (osteogenesisimperfecta); FRAXA syndrome (fragile X syndrome); FRDA (Friedreich'sataxia); Friedreich ataxia (Friedreich's ataxia); Friedreich's ataxia;FXS (fragile X syndrome); G6PD deficiency; Galactokinase deficiencydisease (galactosemia); Galactose-1-phosphate uridyl-transferasedeficiency disease (galactosemia); galactosemia; Galactosylceramidasedeficiency disease (Krabbe disease); Galactosylceramide lipidosis(Krabbe disease); galactosylcerebrosidase deficiency (Krabbe disease);galactosylsphingosine lipidosis (Krabbe disease); GALC deficiency(Krabbe disease); GALT deficiency (galactosemia); Gaucher disease;Gaucher-like disease (pseudo-Gaucher disease); GBA deficiency (Gaucherdisease type 1); GD (Gaucher's disease); Genetic brain disorders;genetic emphysema (alpha-1 antitrypsin deficiency); genetichemochromatosis (hemochromatosis); Giant cell hepatitis, neonatal(Neonatal hemochromatosis); GLA deficiency (Fabry disease);Glioblastoma, retinal (retinoblastoma); Glioma, retinal(retinoblastoma); globoid cell leukodystrophy (GCL, GLD) (Krabbedisease); globoid cell leukoencephalopathy (Krabbe disease);Glucocerebrosidase deficiency (Gaucher disease); Glucocerebrosidosis(Gaucher disease); Glucosyl cerebroside lipidosis (Gaucher disease) ;Glucosylceramidase deficiency (Gaucher disease); Glucosylceramidebeta-glucosidase deficiency (Gaucher disease); Glucosylceramidelipidosis (Gaucher disease); Glyceric aciduria (hyperoxaluria, primary);Glycine encephalopathy (Nonketotic hyperglycinemia); Glycolic aciduria(hyperoxaluria, primary); GM2 gangliosidosis, type 1 (Tay-Sachsdisease); Goiter-deafness syndrome (Pendred syndrome); Graefe-Ushersyndrome (Usher syndrome); Gronblad-Strandberg syndrome (pseudoxanthomaelasticum); Guenther porphyria (congenital erythropoietic porphyria);Gunther disease (congenital erythropoietic porphyria); Haemochromatosis(hemochromatosis); Hallgren syndrome (Usher syndrome); HarlequinIchthyosis; Hb S disease (sickle cell anemia); HCH (hypochondroplasia);HCP (hereditary coproporphyria); Head and brain malformations; Hearingdisorders and deafness; Hearing problems in children; HEF2A(hemochromatosis#type 2); HEF2B (hemochromatosis#type 2);Hematoporphyria (porphyria); Heme synthetase deficiency (erythropoieticprotoporphyria); Hemochromatos es (hem ochromatosi s); hemochromatosis;hemoglobin M disease (methemoglobinemia#beta-globin type); Hemoglobin Sdisease (sickle cell anemia); hemophilia; HEP (hepatoerythropoieticporphyria); hepatic AGT deficiency (hyperoxaluria, primary);hepatoerythropoietic porphyria; Hepatolenticular degeneration syndrome(Wilson disease); Hereditary arthro-ophthalmopathy (Stickler syndrome);Hereditary coproporphyria; Hereditary dystopic lipidosis (Fabrydisease); Hereditary hemochromatosis (HHC) (hemochromatosis); HereditaryInclusion Body Myopathy (skeletal muscle regeneration); Hereditaryiron-loading anemia (X-linked sideroblastic anemia); Hereditary motorand sensory neuropathy (Charcot-Marie-Tooth disease); Hereditary motorneuronopathy (spinal muscular atrophy); Hereditary motor neuronopathy,type V (distal spinal muscular atrophy, type V); Hereditary MultipleExostoses; Hereditary nonpolyposis colorectal cancer; Hereditaryperiodic fever syndrome (Mediterranean fever, familial); HereditaryPolyposis Coli (familial adenomatous polyposis); Hereditary pulmonaryemphysema (alpha-1 antitrypsin deficiency); Hereditary resistance toactivated protein C (factor V Leiden thrombophilia); Hereditary sensoryand autonomic neuropathy type III (familial dysautonomia); Hereditaryspastic paraplegia (infantile-onset ascending hereditary spasticparalysis); Hereditary spinal ataxia (Friedreich ataxia); Hereditaryspinal sclerosis (Friedreich ataxia); Herrick's anemia (sickle cellanemia); Heterozygous OSMED (Wei ss enb acher-Zwey muller syndrome);Heterozygous otospondylomegaepiphyseal dysplasia(Weissenbacher-Zweymuller syndrome); HexA deficiency (Tay-Sachsdisease); Hexosaminidase A deficiency (Tay-Sachs disease);Hexosaminidase alpha-subunit deficiency (variant B) (Tay-Sachs disease);FIFE-associated hemochromatosis (hemochromatosis); HGPS (Progeria);Hippel-Lindau disease (von Hippel-Lindau disease); HLAH(hemochromatosis); EIMN V (distal spinal muscular atrophy, type V);EIMSN (Charcot-Marie-Tooth disease); HNPCC (hereditary nonpolyposiscolorectal cancer); HNPP (hereditary neuropathy with liability topressure palsies); homocystinuria; Homogentisic acid oxidase deficiency(alkaptonuria); Homogentisic acidura (alkaptonuria); Homozygousporphyria cutanea tarda (hepatoerythropoietic porphyria); HP1(hyperoxaluria, primary); HP2 (hyperoxaluria, primary); HPA(hyperphenylalaninemia); HPRT—Hypoxanthine-guaninephosphoribosyltransferase deficiency (Lesch-Nyhan syndrome); HSAN typeIII (familial dysautonomia) ; HSAN3 (familial dysautonomia); HSN-III(familial dysautonomia); Human dermatosparaxis (Ehlers-Danlossyndrome#dermatosparaxis type); Huntington's disease; Hutchinson-Gilfordprogeria syndrome (progeria); Hyperandrogenism, nonclassic type, due to21-hydroxylase deficiency (21-hydroxylase deficiency);Hyperchylomicronemia, familial (lipoprotein lipase deficiency,familial); hyperglycinemia with ketoacidosis and leukopenia (propionicacidemia); Hyperlipoproteinemia type I (lipoprotein lipase deficiency,familial); hyperoxaluria, primary; hyperphenylal aninaemia(hyperphenylalaninemia); hyperphenylalaninemia; Hypochondrodysplasia(hypochondroplasia); hypochondrogenesis; hypochondroplasia; Hypochromicanemia (X-linked sideroblastic anemia); Hypocupremia, congenital; Menkessyndrome); hypoxanthine phosphoribosyltransferse (HPRT) deficiency(Lesch-Nyhan syndrome); IAHSP (infantile-onset ascending hereditaryspastic paralysis); idiopathic hemochromatosis (hemochromatosis, type3); Idiopathic neonatal hemochromatosis (hemochromatosis, neonatal);Idiopathic pulmonary hypertension (primary pulmonary hypertension);Immune system disorders (X-linked severe combined immunodeficiency);Incontinentia Pigmenti; Infantile cerebral Gaucher's disease (Gaucherdisease type 2); Infantile Gaucher disease (Gaucher disease type 2);infantile-onset ascending hereditary spastic paralysis; Infertility;inherited emphysema (alpha-1 antitrypsin deficiency); Inherited humantransmissible spongiform encephalopathies (prion disease); inheritedtendency to pressure palsies (hereditary neuropathy with liability topressure palsies); Insley-Astley syndrome (otospondylomegaepiphysealdysplasia); Intermittent acute porphyria syndrome (acute intermittentporphyria); Intestinal polyposis-cutaneous pigmentation syndrome(Peutz-Jeghers syndrome); IP (incontinentia pigmenti); Iron storagedisorder (hemochromatosis); Isodicentric 15 (idic15); Isolated deafness(nonsyndromic deafness); Jackson-Weiss syndrome; JH(Haemochromatosis#type 2); Joubert syndrome; JPLS (Juvenile PrimaryLateral Sclerosis); juvenile amyotrophic lateral sclerosis (Amyotrophiclateral sclerosis#type 2); Juvenile gout, choreoathetosis, mentalretardation syndrome (Lesch-Nyhan syndrome); juvenile hyperuricemiasyndrome (Lesch-Nyhan syndrome); JWS (Jackson-Weiss syndrome); KD(X-linked spinal-bulbar muscle atrophy); Kennedy disease (X-linkedspinal-bulbar muscle atrophy); Kennedy spinal and bulbar muscularatrophy (X-linked spinal-bulbar muscle atrophy); Kerasin histiocytosis(Gaucher disease); Kerasin lipoidosis (Gaucher disease); Kerasinthesaurismosis (Gaucher disease); ketotic glycinemia (propionicacidemia); ketotic hyperglycinemia (propionic acidemia); Kidney diseases(hyperoxaluria, primary); Klinefelter syndrome; Klinefelter's syndrome;Kniest dysplasia; Krabbe disease; Lacunar dementia (CADASIL);Langer-Saldino achondrogenesis (achondrogenesis, type II);Langer-Saldino dysplasia (achondrogenesis, type II); Late-onsetAlzheimer disease (Alzheimer disease#type 2); Late-onset familialAlzheimer disease (AD2) (Alzheimer disease#type 2); late-onset Krabbedisease (LOKD) (Krabbe disease); Learning Disorders (Learningdisability); Lentiginosis, perioral (Peutz-Jeghers syndrome);Lesch-Nyhan syndrome; Leukodystrophies; leukodystrophy with Rosenthalfibers (Alexander disease); Leukodystrophy, spongiform (Canavandisease); LFS (Li-Fraumeni syndrome); Li-Fraumeni syndrome; Lipase Ddeficiency (lipoprotein lipase deficiency, familial); LIPD deficiency(lipoprotein lipase deficiency, familial); Lipidosis, cerebroside(Gaucher disease); Lipidosis, ganglioside, infantile (Tay-Sachsdisease); Lipoid histiocytosis (kerasin type) (Gaucher disease);lipoprotein lipase deficiency, familial; Liver diseases (galactosemia);Lou Gehrig disease (amyotrophic lateral sclerosis); Louis-Bar syndrome(ataxia-telangiectasia); Lynch syndrome (hereditary nonpolyposiscolorectal cancer); Lysyl-hydroxylase deficiency (Ehlers-Danlossyndrome#kyphoscoliosis type); Machado-Joseph disease (Spinocerebellarataxia#type 3); Male breast cancer (breast cancer); Male genitaldisorders; Male Turner syndrome (Noonan syndrome); Malignant neoplasm ofbreast (breast cancer); malignant tumor of breast (breast cancer);Malignant tumor of urinary bladder (bladder cancer); Mammary cancer(breast cancer); Marfan syndrome 15; Marker X syndrome (fragile Xsyndrome); Martin-Bell syndrome (fragile X syndrome); McCune-Albrightsyndrome; McLeod syndrome; MEDNIK; Mediterranean Anemia (betathalassemia); Mediterranean fever, familial; Mega-epiphyseal dwarfism(otospondylomegaepiphyseal dysplasia); Menkea syndrome (Menkessyndrome); Menkes syndrome; Mental retardation with osteocartilaginousabnormalities (Coffin-Lowry syndrome); Metabolic disorders; Metatropicdwarfism, type II (Kniest dysplasia); Metatropic dysplasia type II(Kniest dysplasia); Methemoglobinemia#beta-globin type; methylmalonicacidemia; MFS (Marfan syndrome); MHAM (Cowden syndrome); MK (Menkessyndrome); Micro syndrome; Microcephaly; MMA (methylmalonic acidemia);MNK (Menkes syndrome); Monosomy 1p36 syndrome (1p36 deletion syndrome);monosomy X (Turner syndrome); Motor neuron disease, amyotrophic lateralsclerosis (amyotrophic lateral sclerosis); Movement disorders;Mowat-Wilson syndrome; Mucopolysaccharidosis (MPS I); Mucoviscidosis(cystic fibrosis); Muenke syndrome; Multi-Infarct dementia (CADASIL);Multiple carboxylase deficiency, late-onset (biotinidase deficiency);Multiple hamartoma syndrome (Cowden syndrome); Multipleneurofibromatosis (neurofibromatosis); Muscular dystrophy; Musculardystrophy, Duchenne and Becker type; Myotonia atrophica (myotonicdystrophy); Myotonia dystrophica (myotonic dystrophy); myotonicdystrophy; Myxedema, congenital (congenital hypothyroidism);Nance-Insley syndrome (otospondylomegaepiphyseal dysplasia);Nance-Sweeney chondrodysplasia (otospondylomegaepiphyseal dysplasia);NBIA1 (pantothenate kinase-associated neurodegeneration); Neill-Dingwallsyndrome (Cockayne syndrome); Neuroblastoma, retinal (retinoblastoma);Neurodegeneration with brain iron accumulation type 1 (pantothenatekinase-associated neurodegeneration); Neurofibromatosis type I;Neurofibromatosis type II; Neurologic diseases; Neuromuscular disorders;neuronopathy, distal hereditary motor, type V (Distal spinal muscularatrophy#type V); neuronopathy, distal hereditary motor, with pyramidalfeatures (Amyotrophic lateral sclerosis#type 4); NF (neurofibromatosis);Niemann-Pick (Niemann-Pick di seas e); Noack syndrome (Pfeiffersyndrome); Nonketotic hyperglycinemia (Glycine encephalopathy);Non-neuronopathic Gaucher disease (Gaucher disease type 1);Non-phenylketonuric hyperphenylalaninemia (tetrahydrobiopterindeficiency); nonsyndromic deafness; Noonan syndrome; NorrbottnianGaucher disease (Gaucher disease type 3); Ochronosis (alkaptonuria);Ochronotic arthritis (alkaptonuria); OI (osteogenesis imperfecta); OSMED(otospondylomegaepiphyseal dysplasia); osteogenesis imperfecta;Osteopsathyrosis (osteogenesis imperfecta); Osteosclerosis congenita(achondroplasia); Oto-spondylo-megaepiphyseal dysplasia(otospondylomegaepiphyseal dysplasia); otospondylomegaepiphysealdysplasia; Oxalosis (hyperoxaluria, primary); Oxaluria, primary(hyperoxaluria, primary); pantothenate kinase-associatedneurodegeneration; Patau Syndrome (Trisomy 13); PBGD deficiency (acuteintermittent porphyria); PCC deficiency (propionic acidemia); PCT(porphyria cutanea tarda); PDM (Myotonic dystrophy#type 2); Pendredsyndrome; Periodic disease (Mediterranean fever, familial); Periodicperitonitis (Mediterranean fever, familial); Periorificial lentiginosissyndrome (Peutz-Jeghers syndrome); Peripheral nerve disorders (familialdysautonomia); Peripheral neurofibromatosis (neurofibromatosis 1);Peroneal muscular atrophy (Charcot-Marie-Tooth disease); peroxisomalalanine:glyoxylate aminotransferase deficiency (hyperoxaluria, primary);Peutz-Jeghers syndrome; Pfeiffer syndrome; Phenylalanine hydroxylasedeficiency disease (phenylketonuria); phenylketonuria; Pheochromocytoma(von Hippel-Lindau disease); Pierre Robin syndrome with fetalchondrodysplasia (Weissenbacher-Zweymuller syndrome); Pigmentarycirrhosis (hemochromatosis); PJS (Peutz-Jeghers syndrome); PKAN(pantothenate kinase-associated neurodegeneration); PKU(phenylketonuria); Plumb op orphyri a (ALA deficiency porphyria); PMA(Charcot-Marie-tooth disease); polyostotic fibrous dysplasia(McCune-Albright syndrome); polyposis coli (familial adenomatouspolyposis); polyposis, hamartomatous intestinal (Peutz-Jegherssyndrome); polyposis, intestinal, II (Peutz-Jeghers syndrome);polyps-and-spots syndrome (Peutz-Jeghers syndrome); Porphobilinogensynthase deficiency (ALA deficiency porphyria); porphyria; porphyrindisorder (porphyria); PPH (primary pulmonary hypertension); PPDXdeficiency (variegate porphyria); Prader-Labhart-Willi syndrome(Prader-Willi syndrome); Prader-Willi syndrome; presenile and seniledementia (Alzheimer disease); primary hemochromatosis (hemochromatosis);primary hyperuricemia syndrome (Lesch-Nyhan syndrome); primary pulmonaryhypertension; primary senile degenerative dementia (Alzheimer disease);prion disease; procollagen type EDS VII, mutant (Ehlers-Danlossyndrome#arthrochalasia type); progeria (Hutchinson Gilford ProgeriaSyndrome); Progeria-like syndrome (Cockayne syndrome); progeroid nanism(Cockayne syndrome); progressive chorea, chronic hereditary (Huntington)(Huntington's disease); progressive muscular atrophy (spinal muscularatrophy); progressively deforming osteogenesis imperfecta with normalsclerae (Osteogenesis imperfecta#type III); PROMM (Myotonicdystrophy#type 2); propionic academia; propionyl-CoA carboxylasedeficiency (propionic acidemia); protein C deficiency; protein Sdeficiency; protoporphyria (erythropoietic protoporphyria);protoporphyrinogen oxidase deficiency (variegate p orphyri a); proximalmyotonic dystrophy (Myotonic dystrophy#type 2); proximal myotonicmyopathy (Myotonic dystrophy#type 2); pseudo-Gaucher disease;pseudo-Ullrich-Turner syndrome (Noonan syndrome); pseudoxanthomaelasticum; psychosine lipidosis (Krabbe disease); pulmonary arterialhypertension (primary pulmonary hypertension); pulmonary hypertension(primary pulmonary hypertension); PWS (Prader-Willi syndrome);PXE—pseudoxanthoma elasticum (pseudoxanthoma elasticum); Rb(retinoblastoma); Recklinghausen disease, nerve (neurofibromatosis 1);Recurrent polyserositis (Mediterranean fever, familial); Retinaldisorders; Retinitis pigmentosa-deafness syndrome (Usher syndrome);Retinoblastoma; Rett syndrome; RFALS type 3 (Amyotrophic lateralsclerosis#type 2); Ricker syndrome (Myotonic dystrophy#type 2);Riley-Day syndrome (familial dysautonomia); Roussy-Levy syndrome(Charcot-Marie-Tooth disease); RSTS (Rubinstein-Taybi syndrome); RTS(Rett syndrome) (Rubinstein-Taybi syndrome); RTT (Rett syndrome);Rubinstein-Taybi syndrome; Sack-Barabas syndrome (Ehlers-Danlossyndrome, vascular type); SADDAN; sarcoma family syndrome of Li andFraumeni (Li-Fraumeni syndrome); sarcoma, breast, leukemia, and adrenalgland (SBLA) syndrome (Li-Fraumeni syndrome); SBLA syndrome (Li-Fraumenisyndrome); SBMA (X-linked spinal-bulbar muscle atrophy); SCD (sicklecell anemia); Schwannoma, acoustic, bilateral (neurofibromatosis 2);SCIDX1 (X-linked severe combined immunodeficiency); sclerosis tuberosa(tuberous sclerosis); SDAT (Alzheimer disease); SED congenita(spondyloepiphyseal dysplasia congenita); SED Strudwick(spondyloepimetaphyseal dysplasia, Strudwick type); SEDc(spondyloepiphyseal dysplasia congenita); SEMD, Strudwick type(spondyloepimetaphyseal dysplasia, Strudwick type); senile dementia(Alzheimer disease#type 2); severe achondroplasia with developmentaldelay and acanthosis nigricans (SADDAN); Shprintzen syndrome (22q11.2deletion syndrome); sickle cell anemia; skeleton-skin-brain syndrome(SADDAN); Skin pigmentation disorders; SMA (spinal muscular atrophy);SMED, Strudwick type (spondyloepimetaphyseal dysplasia, Strudwick type);SMED, type I (spondyloepimetaphyseal dysplasia, Strudwick type); SmithLemli Opitz Syndrome; South-African genetic porphyria (variegateporphyria); spastic paralysis, infantile onset ascending(infantile-onset ascending hereditary spastic paralysis); Speech andcommunication disorders; sphingolipidosis, Tay-Sachs (Tay-Sachsdisease); spinal-bulbar muscular atrophy; spinal muscular atrophy;spinal muscular atrophy, distal type V (Distal spinal muscularatrophy#type V); spinal muscular atrophy, distal, with upper limbpredominance (Distal spinal muscular atrophy#type V); spinocerebellarataxia; spondyloepimetaphyseal dysplasia, Strudwick type;spondyloepiphyseal dysplasia congenital; spondyloepiphyseal dysplasia(collagenopathy, types II and XI); spondylometaepiphyseal dysplasiacongenita, Strudwick type (spondyloepimetaphyseal dysplasia, Strudwicktype); spondylometaphyseal dysplasia (SMD) (spondyloepimetaphysealdysplasia, Strudwick type); spondylometaphyseal dysplasia, Strudwicktype (spondyloepimetaphyseal dysplasia, Strudwick type); spongydegeneration of central nervous system (Canavan disease); spongydegeneration of the brain (Canavan disease); spongy degeneration ofwhite matter in infancy (Canavan disease); sporadic primary pulmonaryhypertension (primary pulmonary hypertension); SSB syndrome (SADDAN);steely hair syndrome (Menkes syndrome); Steinert disease (myotonicdystrophy); Steinert myotonic dystrophy syndrome (myotonic dystrophy);Stickler syndrome; stroke (CADASIL); Strudwick syndrome(spondyloepimetaphyseal dysplasia, Strudwick type); subacuteneuronopathic Gaucher disease (Gaucher disease type 3); Swedish geneticporphyria (acute intermittent porphyria); Swedish porphyria (acuteintermittent porphyria); Swiss cheese cartilage dysplasia (Kniestdysplasia); Tay-Sachs disease; TD—thanatophoric dwarfism (thanatophoricdysplasia); TD with straight femurs and cloverleaf skull (thanatophoricdysplasia#Type 2); Telangiectasia, cerebello-oculocutaneous(ataxia-telangiectasia); Testicular feminization syndrome (androgeninsensitivity syndrome); tetrahydrobiopterin deficiency; TFM—testicularfeminization syndrome (androgen insensitivity syndrome); thalassemiaintermedia (beta thalassemia); Thalassemia Major (beta thalassemia);thanatophoric dysplasia; thiamine-responsive megaloblastic anemia withdiabetes mellitus and sensorineural deafness; Thrombophilia due todeficiency of cofactor for activated protein C, Leiden type (factor VLeiden thrombophilia); Thyroid disease; Tomaculous neuropathy(hereditary neuropathy with liability to pressure palsies); Total HPRTdeficiency (Lesch-Nyhan syndrome); Total hypoxanthine-guaninephosphoribosyl transferase deficiency (Lesch-Nyhan syndrome); Tourette'sSyndrome; Transmissible dementias (prion disease); Transmissiblespongiform encephalopathies (prion disease); Treacher Collins syndrome;Trias fragilitis ossium (osteogenesis imperfecta#Type I); triple Xsyndrome; Triplo X syndrome (triple X syndrome); Trisomy 21 (Downsyndrome); Trisomy X (triple X syndrome); Troisier-Hanot-Chauffardsyndrome (hemochromatosis); TS (Turner syndrome); TSD (Tay-Sachsdisease); TSEs (prion disease); tuberose sclerosis (tuberous sclerosis);tuberous sclerosis; Turner syndrome; Turner syndrome in female with Xchromosome (Noonan syndrome); Turner's phenotype, karyotype normal(Noonan syndrome); Turner's syndrome (Turner syndrome); Turner-likesyndrome (Noonan syndrome); Type 2 Gaucher disease (Gaucher disease type2); Type 3 Gaucher disease (Gaucher disease type 3);UDP-galactose-4-epimerase deficiency disease (galactosemia); UDP glucose4-epimerase deficiency disease (galactosemia); UDP glucosehexose-1-phosphate uridylyltransferase deficiency (galactosemia);Ullrich-Noonan syndrome (Noonan syndrome); Ullrich-Turner syndrome(Turner syndrome); Undifferentiated deafness (nonsyndromic deafness);UPS deficiency (acute intermittent porphyria); Urinary bladder cancer(bladder cancer); UROD deficiency (porphyria cutanea tarda);Uroporphyrinogen decarboxylase deficiency (porphyria cutanea tarda);Uroporphyrinogen synthase deficiency (acute intermittent porphyria);UROS deficiency (congenital erythropoietic porphyria); Usher syndrome;UTP hexose-l-phosphate uridylyltransferase deficiency (galactosemia);Van Bogaert-Bertrand syndrome (Canavan disease); Van der Hoeve syndrome(osteogenesis imperfecta#Type I); variegate porphyria; Velocardiofacialsyndrome (22q11.2 deletion syndrome); VHL syndrome (von Hippel-Lindaudisease); Vision impairment and blindness (Alstrom syndrome); VonBogaert-Bertrand disease (Canavan disease); von Hippel-Lindau disease;Von Recklenhausen-Applebaum disease (hemochromatosis); vonRecklinghausen disease (neurofibromatosis 1); VP (variegate porphyria);Vrolik disease (osteogenesis imperfecta); Waardenburg syndrome; WarburgSjo Fledelius Syndrome (Micro syndrome); WD (Wilson disease);Weissenbacher-Zweymtiller syndrome; Wilson disease; Wilson's disease(Wilson disease); Wolf-Hirschhorn syndrome; Wolff Periodic disease(Mediterranean fever, familial); WZS (Weissenbacher-Zweymtillersyndrome); Xeroderma Pigmentosum; X-linked mental retardation andmacroorchidism (fragile X syndrome); X-linked primary hyperuricemia(Lesch-Nyhan syndrome); X-linked severe combined immunodeficiency;X-linked sideroblastic anemia; X-linked spinal-bulbar muscle atrophy(Kennedy disease); X-linked uric aciduria enzyme defect (Lesch-Nyhansyndrome); X-SCID (X-linked severe combined immunodeficiency); XLSA(X-linked sideroblastic anemia); XSCID (X-linked severe combinedimmunodeficiency); XXX syndrome (triple X syndrome); XXXX syndrome (48,XXXX); XXXXX syndrome (49, XXXXX); XXY syndrome (Klinefelter syndrome);XXY trisomy (Klinefelter syndrome); XYY karyotype (47,XYY syndrome); XYYsyndrome (47,XYY syndrome); and YY syndrome (47,XYY syndrome).

In a further preferred aspect, the inventive nucleic acid sequence asdefined herein or the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein may be used for thepreparation of a pharmaceutical composition, particularly for purposesas defined herein, preferably for the use in gene therapy in thetreatment of diseases as defined herein.

The inventive pharmaceutical composition may furthermore be used in genetherapy particularly in the treatment of a disease or a disorder,preferably as defined herein.

According to a final aspect, the present invention also provides kits,particularly kits of parts. Such kits, particularly kits of parts,typically comprise as components alone or in combination with furthercomponents as defined herein at least one inventive nucleic acidsequence as defined herein, the inventive pharmaceutical composition orvaccine comprising the inventive nucleic acid sequence. The at least oneinventive nucleic acid sequence as defined herein, is optionally incombination with further components as defined herein, whereby the atleast one nucleic acid of the invention is provided separately (firstpart of the kit) from at least one other part of the kit comprising oneor more other components. The inventive pharmaceutical composition maye.g. occur in one or different parts of the kit. As an example, e.g. atleast one part of the kit may comprise at least one inventive nucleicacid sequence as defined herein, and at least one further part of thekit at least one other component as defined herein, e.g. at least oneother part of the kit may comprise at least one pharmaceuticalcomposition or a part thereof, e.g. at least one part of the kit maycomprise the inventive nucleic acid sequence as defined herein, at leastone further part of the kit at least one other component as definedherein, at least one further part of the kit at least one component ofthe inventive pharmaceutical composition or the inventive pharmaceuticalcomposition as a whole, and at least one further part of the kit e.g. atleast one pharmaceutical carrier or vehicle, etc. In case the kit or kitof parts comprises a plurality of inventive nucleic acid sequences, onecomponent of the kit can comprise only one, several or all inventivenucleic acid sequences comprised in the kit. In an alternativeembodiment every/each inventive nucleic acid sequence may be comprisedin a different/separate component of the kit such that each componentforms a part of the kit. Also, more than one nucleic acid may becomprised in a first component as part of the kit, whereas one or moreother (second, third etc.) components (providing one or more other partsof the kit) may either contain one or more than one inventive nucleicacids, which may be identical or partially identical or different fromthe first component. The kit or kit of parts may furthermore containtechnical instructions with information on the administration and dosageof the inventive nucleic acid sequence, the inventive pharmaceuticalcomposition or of any of its components or parts, e.g. if the kit isprepared as a kit of parts.

Taken together, the invention provides nucleic acid sequence comprisingor coding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, may be a        therapeutic protein,    -   preferably a therapeutic protein for use in the treatment of        metabolic or endocrine disorders, for use in the treatment of        blood disorders, diseases of the circulatory system, diseases of        the respiratory system, cancer or tumour diseases, infectious        diseases or immunedeficiencies, for use in hormone replacement        therapy or for use in reprogramming of somatic cells    -   or a therapeutic protein selected from from adjuvant or        immunostimulating proteins, human adjuvant proteins, bacterial        (adjuvant) proteins, protozoan (adjuvant) proteins, viral        (adjuvant) proteins, fungal (adjuvant) proteins    -   or an antibody, preferably an antibody selected from antibodies        used for the treatment of cancer or tumour diseases, antibodies        used for the treatment of immune disorders, antibodies used for        the treatment of infectious diseases, antibodies used for the        treatment of infectious diseases, antibodies used for the        treatment of blood disorders, antibodies used for        immunoregulation, antibodies used for the treatment of diabetes,        antibodies used for the treatment of the Alzheimer's disease,        antibodies used for the treatment of asthma or antibodies for        the treatment of diverse disorders.    -   The invention further provides the use of such a nucleic acid        sequence in gene therapy as defined herein. The invention        further provides a kit or kit of parts comprising such a nucleic        acid sequence. Further, the invention provides a pharmaceutical        composition comprising such a nucleic acid sequence. Further,        the invention provides a method for increasing the expression of        an encoded peptide or protein comprising the steps of providing        such a nucleic acid sequence or a composition containing such a        nucleic acid sequence and applying or administering the nucleic        acid sequence or othe composition to a cell-free expression        system, a cell, a tissue or an organism.

Preferably, the invention provides a nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, may be a        therapeutic protein        -   for use in the treatment of metabolic or endocrine            disorders,        -   for use in the treatment of blood disorders, diseases of the            circulatory system, diseases of the respiratory system,            cancer or tumour diseases, infectious diseases or            immunedeficiencies,        -   for use in hormone replacement therapy or        -   for use in reprogramming of somatic cells.    -   The invention further provides the use of such a nucleic acid        sequence in gene therapy as defined herein. The invention        further provides a kit or kit of parts comprising such a nucleic        acid sequence. Further, the invention provides a pharmaceutical        composition comprising such a nucleic acid sequence. Further,        the invention provides a method for increasing the expression of        an encoded peptide or protein comprising the steps of providing        such a nucleic acid sequence or a composition containing such a        nucleic acid sequence and applying or administering the nucleic        acid sequence or othe composition to a cell-free expression        system, a cell, a tissue or an organism.

Further preferably, the invention provides a nucleic acid sequencecomprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein selected from adjuvant or immunostimulating        proteins, more preferably selected from human adjuvant proteins,        bacterial (adjuvant) proteins, protozoan (adjuvant) proteins,        viral (adjuvant) proteins, fungal (adjuvant) proteins.    -   The invention further provides the use of such a nucleic acid        sequence in gene therapy as defined herein. The invention        further provides a kit or kit of parts comprising such a nucleic        acid sequence. Further, the invention provides a pharmaceutical        composition comprising such a nucleic acid sequence. Further,        the invention provides a method for increasing the expression of        an encoded peptide or protein comprising the steps of providing        such a nucleic acid sequence or a composition containing such a        nucleic acid sequence and applying or administering the nucleic        acid sequence or othe composition to a cell-free expression        system, a cell, a tissue or an organism.

Further preferably, the invention provides a nucleic acid sequencecomprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein or a fragment, variant or derivative thereof        which is a therapeutic antibody, more preferably an antibody        selected from antibodies used for the treatment of cancer or        tumour diseases, antibodies used for the treatment of immune        disorders, antibodies used for the treatment of infectious        diseases, antibodies used for the treatment of infectious        diseases, antibodies used for the treatment of blood disorders,        antibodies used for immunoregulation, antibodies used for the        treatment of diabetes, antibodies used for the treatment of the        Alzheimer's disease, antibodies used for the treatment of asthma        or antibodies for the treatment of diverse disorders.    -   The invention further provides the use of such a nucleic acid        sequence in gene therapy as defined herein. The invention        further provides a kit or kit of parts comprising such a nucleic        acid sequence. Further, the invention provides a pharmaceutical        composition comprising such a nucleic acid sequence. Further,        the invention provides a method for increasing the expression of        an encoded peptide or protein comprising the steps of providing        such a nucleic acid sequence or a composition containing such a        nucleic acid sequence and applying or administering the nucleic        acid sequence or othe composition to a cell-free expression        system, a cell, a tissue or an organism.

Preferably the invention provides a nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein for use in the treatment of metabolic or        endocrine disorders,        -   more preferably a peptide or protein selected from Acid            sphingomyelinase, Adipotide, Agalsidase-beta, Alglucosidase,            alpha-galactosidase A, alpha-glucosidase,            alpha-L-iduronidase, alpha-N-acetylglucosaminidase,            Amphiregulin, Angiopoietins (Ang1, Ang2, Ang3, Ang4,            ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7),            Betacellulin, Beta-glucuronidase, Bone morphogenetic            proteins BMPs (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7,            BMP8a, BMP8b, BMP10, BMP15), CLN6 protein, Epidermal growth            factor (EGF), Epigen, Epiregulin, Fibroblast Growth Factor            (FGF, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7,            FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14,            FGF-16, FGF-17, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21,            FGF-22, FGF-23), Galsulphase, Ghrelin, Glucocerebrosidase,            GM-CSF, Heparin-binding EGF-like growth factor (HB-EGF),            Hepatocyte growth factor HGF, Hepcidin, Human albumin,            increased loss of albumin, Idursulphase            (Iduronate-2-sulphatase), Integrins αVβ3, αVβ5 and α5β1,            Iuduronate sulfatase, Laronidase,            N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase,            Arylsulfatase A (ARSA), Arylsulfatase B (ARSB)),            N-acetylglucosamine-6-sulfatase, Nerve growth factor (NGF,            Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3            (NT-3), and Neurotrophin 4/5 (NT-4/5), Neuregulin (NRG1,            NRG2, NRG3, NRG4), Neuropilin (NRP-1, NRP-2), Obestatin,            Platelet Derived Growth factor (PDGF (PDFF-A, PDGF-B,            PDGF-C, PDGF-D), TGF beta receptors (endoglin, TGF-beta 1            receptor, TGF-beta 2 receptor, TGF-beta 3 receptor),            Thrombopoietin (THPO) (Megakaryocyte growth and development            factor (MGDF)), Transforming Growth factor (TGF (TGF-a,            TGF-beta (TGFbeta1, TGFbeta2, and TGFbeta3))), VEGF (VEGF-A,            VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PIGF),            Nesiritide, Trypsin, adrenocorticotrophic hormone (ACTH),            Atrial-natriuretic peptide (ANP), Cholecystokinin, Gastrin,            Leptin, Oxytocin, Somatostatin, Vasopressin (antidiuretic            hormone), Calcitonin, Exenatide, Growth hormone (GH),            somatotropin, Insulin, Insulin-like growth factor 1 IGF-1,            Mecasermin rinfabate, IGF-1 analog, Mecasermin, IGF-1            analog, Pegvisomant, Pramlintide, Teriparatide (human            parathyroid hormone residues 1-34), Becaplermin,            Dibotermin-alpha (Bone morphogenetic protein 2), Histrelin            acetate (gonadotropin releasing hormone; GnRH), Octreotide,            and Palifermin (keratinocyte growth factor; KGF).

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein for use in the treatment of blood disorders,        diseases of the circulatory system, diseases of the respiratory        system, cancer or tumour diseases, infectious diseases or        immunedeficiencies, more preferably a peptide or protein        selected from Alteplase (tissue plasminogen activator; tPA),        Anistreplase, Antithrombin III (AT-III), Bivalirudin,        Darbepoetin-alpha, Drotrecogin-alpha (activated protein C,        Erythropoietin, Epoetin-alpha, erythropoetin, erthropoyetin,        Factor IX, Factor VIIa, Factor VIII, Lepirudin, Protein C        concentrate, Reteplase (deletion mutein of tPA), Streptokinase,        Tenecteplase, Urokinase, Angiostatin, Anti-CD22 immunotoxin,        Denileukin diftitox, Immunocyanin, MPS (Metallopanstimulin),        Aflibercept, Endostatin, Collagenase, Human deoxy-ribonuclease        I, dornase, Hyaluronidase, Papain, L-Asparaginase,        Peg-asparaginase, Rasburicase, Human chorionic gonadotropin        (HCG), Human follicle-stimulating hormone (FSH), Lutropin-alpha,        Prolactin, alpha-1-Proteinase inhibitor, Lactase, Pancreatic        enzymes (lipase, amylase, protease), Adenosine deaminase        (pegademase bovine, PEG-ADA), Abatacept, Alefacept, Anakinra,        Etanercept, Interleukin-1 (IL-1) receptor antagonist, Anakinra,        Thymulin, TNF-alpha antagonist, Enfuvirtide, and Thymosin α1.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein used for hormone replacement therapy, more        preferably a peptide or protein selected from oestrogens,        progesterone or progestins, and testosterone.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.Preferably the inventionprovides nucleic acid sequence comprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;        -   wherein said peptide or protein comprises a therapeutic            protein or a fragment, variant or derivative thereof,            preferably a therapeutic protein used for reprogramming of            somatic cells into pluri- or omnipotent stem cells, more            preferably a peptide or protein selected from Oct-3/4, Sox            gene family (Sox1, Sox2, Sox3, and Sox15), Klf family (Klf1,            Klf2, Klf4, and Klf5), Myc family (c-myc, L-myc, and N-myc),            Nanog, and LIN28.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.Preferably the inventionprovides nucleic acid sequence comprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein selected from adjuvant or immunostimulating        proteins, human adjuvant proteins, bacterial (adjuvant)        proteins, protozoan (adjuvant) proteins, viral (adjuvant)        proteins, fungal (adjuvant) proteins.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein selected from adjuvant or immunostimulating        proteins, human adjuvant proteins, bacterial (adjuvant)        proteins, protozoan (adjuvant) proteins, viral (adjuvant)        proteins, fungal (adjuvant) proteins, more preferably selected        from human adjuvant proteins, most preferably selected from        pattern recognition receptors TLR1, TLR2, TLR3, TLR4, TLR5,        TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NOD1, NOD2, NOD3, NOD4,        NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7,        NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14,1        IPAF, NAIP, CIITA, RIG-I, MDAS and LGP2, the signal transducers        of TLR signaling including adaptor proteins including e.g. Trif        and Cardif; components of the Small-GTPases signalling (RhoA,        Ras, Rac1, Cdc42, Rab etc.), components of the PIP signalling        (PI3K, Src-Kinases, etc.), components of the MyD88-dependent        signalling (MyD88, IRAK1, IRAK2, IRAK4, TIRAP, TRAF6 etc.),        components of the MyD88-independent signalling (TICAM1, TICAM2,        TRAF6, TBK1, IRF3, TAK1, IRAK1 etc.); the activated kinases        including e.g. Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1,        ERK2, GSK3, PKC kinases, PKD kinases, GSK3 kinases, JNK,        p38MAPK, TAK1, IKK, and TAK1; the activated transcription        factors including e.g. NF-κB, c-Fos, c-Jun, c-Myc, CREB, AP-1,        Elk-1, ATF2, IRF-3, IRF-7, heat shock proteins, such as HSP10,        HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIII        repeat extra domain A of fibronectin; or components of the        complement system including C1q, MBL, C1r, C1s, C2b, Bb, D,        MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9,        CR1, CR2, CR3, CR4, C1qR, C1INH, C4bp, MCP, DAF, H, I, P and        CD59, or induced target genes including e.g. Beta-Defensin, cell        surface proteins; or human adjuvant proteins including trif,        flt-3 ligand, Gp96 or fibronectin, cytokines which induce or        enhance an innate immune response, including IL-1 alpha, IL1        beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, IL-15, IL-16,        IL-17, IL-18, IL-21, IL-23, TNFalpha, IFNalpha, IFNbeta,        IFNgamma, GM-CSF, G-CSF, M-CSF; chemokines including IL-8,        IP-10, MCP-1, MIP-1alpha, RANTES, Eotaxin, CCL21; cytokines        which are released from macrophages, including IL-1, IL-6, IL-8,        IL-12 and TNF-alpha; as well as IL-1R1 and IL-1 alpha.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein selected from adjuvant or immunostimulating        proteins, human adjuvant proteins, bacterial (adjuvant)        proteins, protozoan (adjuvant) proteins, viral (adjuvant)        proteins, fungal (adjuvant) proteins, more preferably selected        from bacterial (adjuvant) proteins, most preferably selected        from bacterial heat shock proteins or chaperons, including        Hsp60, Hsp70, Hsp90, Hsp 100; OmpA (Outer membrane protein) from        gram-negative bacteria; bacterial porins, including OmpF;        bacterial toxins, including pertussis toxin (PT) from Bordetella        pertussis, pertussis adenylate cyclase toxin CyaA and CyaC from        Bordetella pertussis, PT-9K/129G mutant from pertussis toxin,        pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella        pertussis, tetanus toxin, cholera toxin (CT), cholera toxin        B-subunit, CTK63 mutant from cholera toxin, CTE112K mutant from        CT, Escherichia coli heat-labile enterotoxin (LT), B subunit        from heat-labile enterotoxin (LTB) Escherichia coli heat-labile        enterotoxin mutants with reduced toxicity, including LTK63,        LTR72; phenol-soluble modulin; neutrophil-activating protein        (HP-NAP) from Helicobacter pylori; Surfactant protein D; Outer        surface protein A lipoprotein from Borrelia burgdorferi, Ag38        (38 kDa antigen) from Mycobacterium tuberculosis; proteins from        bacterial fimbriae; Enterotoxin CT of Vibrio cholerae, Pilin        from pili from gram negative bacteria, and Surfactant protein A        and bacterial flagellins.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein selected from adjuvant or immunostimulating        proteins, human adjuvant proteins, bacterial (adjuvant)        proteins, protozoan (adjuvant) proteins, viral (adjuvant)        proteins, fungal (adjuvant) proteins, more preferably selected        from protozoan (adjuvant) proteins, most preferably selected        from Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii,        Protozoan heat shock proteins, LeIF from Leishmania spp.,        profiling-like protein from Toxoplasma gondii.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein selected from adjuvant or immunostimulating        proteins, human adjuvant proteins, bacterial (adjuvant)        proteins, protozoan (adjuvant) proteins, viral (adjuvant)        proteins, fungal (adjuvant) proteins, more preferably selected        from viral (adjuvant) proteins, most preferably selected from        Respiratory Syncytial Virus fusion glycoprotein (F-protein),        envelope protein from MMT virus, mouse leukemia virus protein,        Hemagglutinin protein of wild-type measles virus.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein selected from adjuvant or immunostimulating        proteins, human adjuvant proteins, bacterial (adjuvant)        proteins, protozoan (adjuvant) proteins, viral (adjuvant)        proteins, fungal (adjuvant) proteins, more preferably selected        from fungal (adjuvant) proteins, most preferably selected from        fungal immunomodulatory protein (FIP; LZ-8); and Keyhole limpet        hemocyanin (KLH), OspA.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or other composition to a cell-free expressionsystem, a cell, a tissue or an organism. Preferably the inventionprovides nucleic acid sequence comprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic antibody selected from antibodies used for the        treatment of cancer or tumour diseases, preferably        131I-tositumomab, 3F8, 8H9, Abagovomab, Adecatumumab,        Afutuzumab, Alacizumab pegol, Alemtuzumab, Amatuximab, AME-133v,        AMG 102, Anatumomab mafenatox, Apolizumab, Bavituximab,        Bectumomab, Belimumab, Bevacizumab, Bivatuzumab mertansine,        Blinatumomab, Brentuximab vedotin, Cantuzumab, Cantuzumab        mertansine, Cantuzumab ravtansine, Capromab pendetide, Carlumab,        Catumaxomab, Cetuximab, Citatuzumab bogatox, Cixutumumab,        Clivatuzumab tetraxetan, CNTO 328, CNTO 95, Conatumumab,        Dacetuzumab, Dalotuzumab, Denosumab, Detumomab, Drozitumab,        Ecromeximab, Edrecolomab, Elotuzumab, Elsilimomab, Enavatuzumab,        Ensituximab, Epratuzumab, Ertumaxomab, Ertumaxomab,        Etaracizumab, Farletuzumab, FBTA05, Ficlatuzumab, Figitumumab,        Flanvotumab, Galiximab, Galiximab, Ganitumab, GC1008,        Gemtuzumab, Gemtuzumab ozogamicin, Girentuximab, Glembatumumab        vedotin, GS6624, HuC242-DM4, HuHMFG1, HuN901-DM1, Ibritumomab,        Icrucumab, ID09C3, Indatuximab ravtansine, Inotuzumab        ozogamicin, Intetumumab, Ipilimumab, Iratumumab, Labetuzumab,        Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab,        Lumiliximab, Mapatumumab, Matuzumab, MDX-060, MEDI 522,        Mitumomab, Mogamulizumab, MORab-003, MORab-009, Moxetumomab        pasudotox, MT103, Nacolomab tafenatox, Naptumomab estafenatox,        Narnatumab, Necitumumab, Nimotuzumab, Nimotuzumab, Olaratumab,        Onartuzumab, Oportuzumab monatox, Oregovomab, Oregovomab, PAM4,        Panitumumab, Patritumab, Pemtumomab, Pertuzumab, Pritumumab,        Racotumomab, Radretumab, Ramucirumab, Rilotumumab, Rituximab,        Robatumumab, Samalizumab, SGN-30, SGN-40, Sibrotuzumab,        Siltuximab, Tabalumab, Tacatuzumab tetraxetan, Taplitumomab        paptox, Tenatumomab, Teprotumumab, TGN1412, Ticilimumab,        Tigatuzumab, TNX-650, Tositumomab, Trastuzumab, TRBS07,        Tremelimumab, TRU-016, TRU-016, Tucotuzumab celmoleukin,        Ublituximab, Urelumab, Veltuzumab, Veltuzumab (IMMU-106),        Volociximab, Votumumab, WX-G250, Zalutumumab, and Zanolimumab.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic antibody selected from antibodies used for the        treatment of immune disorders, preferably selected from        Efalizumab, Epratuzumab, Etrolizumab, Fontolizumab, Ixekizumab,        Mepolizumab, Milatuzumab, Pooled immunoglobulins, Priliximab,        Rituximab, Rontalizumab, Ruplizumab, Sarilumab, Vedolizumab,        Visilizumab, Reslizumab, Adalimumab, Aselizumab, Atinumab,        Atlizumab, Bertilimumab, Besilesomab, BMS-945429, Briakinumab,        Brodalumab, Canakinumab, Canakinumab, Certolizumab pegol,        Erlizumab, Fezakinumab, Golimumab, Gomiliximab, Infliximab,        Mavrilimumab, Natalizumab, Ocrelizumab, Odulimomab, Ofatumumab,        Ozoralizumab, Pexelizumab, Rovelizumab, SBI-087, SBI-087,        Secukinumab, Sirukumab, Talizumab, Tocilizumab, Toralizumab,        TRU-015, TRU-016, Ustekinumab, Ustekinumab, Vepalimomab,        Zolimomab aritox, Sifalimumab, Lumiliximab, and Rho(D) Immune        Globulin.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic antibody selected from antibodies used for the        treatment of cancer or tumour diseases, preferably antibodies        used for the treatment of infectious diseases, particularly        Afelimomab, CR6261, Edobacomab, Efungumab, Exbivirumab,        Felvizumab, Foravirumab, Ibalizumab, Libivirumab, Motavizumab,        Nebacumab, Tuvirumab, Urtoxazumab, Bavituximab, Pagibaximab,        Palivizumab, Panobacumab, PRO 140, Rafivirumab, Raxibacumab,        Regavirumab, Sevirumab, Suvizumab, and Tefibazumab.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic antibody selected from antibodies used for the        treatment of cancer or tumour diseases, preferably antibodies        used for the treatment of blood disorders, particularly        Abciximab, Atorolimumab, Eculizumab, Mepolizumab, and        Milatuzumab.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic antibody selected from antibodies used for the        treatment of cancer or tumour diseases, preferably antibodies        used for immunoregulation, particularly Antithymocyte globulin,        Basiliximab, Cedelizumab, Daclizumab, Gavilimomab, Inolimomab,        Muromonab-CD3, Muromonab-CD3, Odulimomab, and Siplizumab.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic antibody selected from antibodies used for the        treatment of cancer or tumour diseases, preferably antibodies        used for the treatment of diabetes, particularly Gevokizumab,        Otelixizumab, and Teplizumab.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic antibody selected from antibodies used for the        treatment of cancer or tumour diseases, preferably antibodies        used for the treatment of the Alzheimer's disease, particularly        Bapineuzumab, Crenezumab, Gantenerumab, Ponezumab, R1450, and        Solanezumab.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic antibody selected from antibodies used for the        treatment of cancer or tumour diseases, preferably antibodies        used for the treatment of asthma, particularly Benralizumab,        Enokizumab, Keliximab, Lebrikizumab, Omalizumab, Oxelumab,        Pascolizumab, and Tralokinumab.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic antibody selected from antibodies used for the        treatment of cancer or tumour diseases, preferably antibodies        for the treatment of diverse disorders, particularly Blosozumab,        CaroRx, Fresolimumab, Fulranumab, Romosozumab, Stamulumab,        Tanezumab, and Ranibizumab.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably, the invention provides a nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, may be a        therapeutic protein        -   for use in the treatment of metabolic or endocrine            disorders,        -   for use in the treatment of blood disorders, diseases of the            circulatory system, diseases of the respiratory system,            cancer or tumour diseases, infectious diseases or            immunedeficiencies,        -   for use in hormone replacement therapy or        -   for use in reprogramming of somatic cells        -   more preferably a therapeutic protein selected from Acid            sphingomyelinase, Adipotide, Agalsidase-beta, Alglucosidase,            alpha-galactosidase A, alpha-glucosidase,            alpha-L-iduronidase, alpha-N-acetylglucosaminidase,            Amphiregulin, Angiopoietins (Ang1, Ang2, Ang3, Ang4,            ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7),            Betacellulin, Beta-glucuronidase, Bone morphogenetic            proteins BMPs (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7,            BMP8a, BMP8b, BMP10, BMP15), CLN6 protein, Epidermal growth            factor (EGF), Epigen, Epiregulin, Fibroblast Growth Factor            (FGF, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7,            FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14,            FGF-16, FGF-17, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21,            FGF-22, FGF-23), Galsulphase, Ghrelin, Glucocerebrosidase,            GM-CSF, Heparin-binding EGF-like growth factor (HB-EGF),            Hepatocyte growth factor HGF, Hepcidin, Human albumin,            increased loss of albumin, Idursulphase            (Iduronate-2-sulphatase), Integrins αVβ3, αVβ5 and α5β1,            Iuduronate sulfatase, Laronidase,            N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase,            Arylsulfatase A (ARSA), Arylsulfatase B (ARSB)),            N-acetylglucosamine-6-sulfatase, Nerve growth factor (NGF,            Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3            (NT-3), and Neurotrophin 4/5 (NT-4/5), Neuregulin (NRG1,            NRG2, NRG3, NRG4), Neuropilin (NRP-1, NRP-2), Obestatin,            Platelet Derived Growth factor (PDGF (PDFF-A, PDGF-B,            PDGF-C, PDGF-D), TGF beta receptors (endoglin, TGF-beta 1            receptor, TGF-beta 2 receptor, TGF-beta 3 receptor),            Thrombopoietin (THPO) (Megakaryocyte growth and development            factor (MGDF)), Transforming Growth factor (TGF (TGF-a,            TGF-beta (TGFbeta1, TGFbeta2, and TGFbeta3))), VEGF (VEGF-A,            VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PIGF),            Nesiritide, Trypsin, adrenocorticotrophic hormone (ACTH),            Atrial-natriuretic peptide (ANP), Cholecystokinin, Gastrin,            Leptin, Oxytocin, Somatostatin, Vasopressin (antidiuretic            hormone), Calcitonin, Exenatide, Growth hormone (GH),            somatotropin, Insulin, Insulin-like growth factor 1 IGF-1,            Mecasermin rinfab ate, IGF-1 analog, Mecasermin, IGF-1            analog, Pegvisomant, Pramlintide, Teriparatide (human            parathyroid hormone residues 1-34), B ecaplermin,            Dibotermin-alpha (Bone morphogenetic protein 2), Histrelin            acetate (gonadotropin releasing hormone; GnRH), Octreotide,            and Palifermin (keratinocyte growth factor; KGF), Alteplase            (tissue plasminogen activator; tPA), Anistreplase,            Antithrombin III (AT-III), Bivalirudin, Darbepoetin-alpha,            Drotrecogin-alpha (activated protein C, Erythropoietin,            Epoetin-alpha, erythropoetin, erthropoyetin, Factor IX,            Factor VIIa, Factor VIII, Lepirudin, Protein C concentrate,            Reteplase (deletion mutein of tPA), Streptokinase,            Tenecteplase, Urokinase, Angiostatin, Anti-CD22 immunotoxin,            Denileukin diftitox, Immunocyanin, MPS (Metallopanstimulin),            Aflibercept, Endostatin, Collagenase, Human            deoxy-ribonuclease I, dornase, Hyaluronidase, Papain,            L-Asparaginase, Peg-asparaginase, Rasburicase, Human            chorionic gonadotropin (HCG), Human follicle-stimulating            hormone (FSH), Lutropin-alpha, Prolactin, alpha-1-Proteinase            inhibitor, Lactase, Pancreatic enzymes (lipase, amylase,            protease), Adenosine deaminase (pegademase bovine, PEG-ADA),            Abatacept, Alefacept, Anakinra, Etanercept, Interleukin-1            (IL-1) receptor antagonist, Anakinra, Thymulin, TNF-alpha            antagonist, Enfuvirtide, Thymosin α1, oestrogens,            progesterone or progestins, testosterone, Oct-3/4, Sox gene            family (Sox1, Sox2, Sox3, and Sox15), Klf family (Klf1,            Klf2, Klf4, and Klf5), Myc family (c-myc, L-myc, and N-myc),            Nanog, and LIN28.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein selected from adjuvant or immunostimulating        proteins, human adjuvant proteins, bacterial (adjuvant)        proteins, protozoan (adjuvant) proteins, viral (adjuvant)        proteins, fungal (adjuvant) proteins, more preferably selected        from human adjuvant proteins, most preferably selected from        pattern recognition receptors TLR1, TLR2, TLR3, TLR4, TLR5,        TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NOD1, NOD2, NOD3, NOD4,        NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7,        NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14,1        IPAF, NAIP, CIITA, RIG-I, MDAS and LGP2, the signal transducers        of TLR signaling including adaptor proteins including e.g. Trif        and Cardif; components of the Small-GTPases signalling (RhoA,        Ras, Rac1, Cdc42, Rab etc.), components of the PIP signalling        (PI3K, Src-Kinases, etc.), components of the MyD88-dependent        signalling (MyD88, IRAK1, IRAK2, IRAK4, TIRAP, TRAF6 etc.),        components of the MyD88-independent signalling (TICAM1, TICAM2,        TRAF6, TBK1, IRF3, TAK1, IRAK1 etc.); the activated kinases        including e.g. Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1,        ERK2, GSK3, PKC kinases, PKD kinases, GSK3 kinases, JNK,        p38MAPK, TAK1, IKK, and TAK1; the activated transcription        factors including e.g. NF-κB, c-Fos, c-Jun, c-Myc, CREB, AP-1,        Elk-1, ATF2, IRF-3, IRF-7, heat shock proteins, such as HSP10,        HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIII        repeat extra domain A of fibronectin; or components of the        complement system including C1q, MBL, C1r, C1s, C2b, Bb, D,        MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9,        CR1, CR2, CR3, CR4, C1qR, ClINH, C4bp, MCP, DAF, H, I, P and        CD59, or induced target genes including e.g. Beta-Defensin, cell        surface proteins; or human adjuvant proteins including trif,        flt-3 ligand, Gp96 or fibronectin, cytokines which induce or        enhance an innate immune response, including IL-1 alpha, IL1        beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, IL-15, IL-16,        IL-17, IL-18, IL-21, IL-23, TNFalpha, IFNalpha, IFNbeta,        IFNgamma, GM-CSF, G-CSF, M-CSF; chemokines including IL-8,        IP-10, MCP-1, MIP-1alpha, RANTES, Eotaxin, CCL21; cytokines        which are released from macrophages, including IL-1, IL-6, IL-8,        IL-12 and TNF-alpha; as well as IL-1R1 and IL-1 alpha, bacterial        heat shock proteins or chaperons, including Hsp60, Hsp70, Hsp90,        Hsp100; OmpA (Outer membrane protein) from gram-negative        bacteria; bacterial porins, including OmpF; bacterial toxins,        including pertussis toxin (PT) from Bordetella pertussis,        pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella        pertussis, PT-9K/129G mutant from pertussis toxin, pertussis        adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis,        tetanus toxin, cholera toxin (CT), cholera toxin B-subunit,        CTK63 mutant from cholera toxin, CTE112K mutant from CT,        Escherichia coli heat-labile enterotoxin (LT), B subunit from        heat-labile enterotoxin (LTB) Escherichia coli heat-labile        enterotoxin mutants with reduced toxicity, including LTK63,        LTR72; phenol-soluble modulin; neutrophil-activating protein        (HP-NAP) from Helicobacter pylori; Surfactant protein D; Outer        surface protein A lipoprotein from Borrelia burgdorferi, Ag38        (38 kDa antigen) from Mycobacterium tuberculosis; proteins from        bacterial fimbriae; Enterotoxin CT of Vibrio cholerae, Pilin        from pili from gram negative bacteria, and Surfactant protein A        and bacterial flagellins, Tc52 from Trypanosoma cruzi, PFTG from        Trypanosoma gondii, Protozoan heat shock proteins, LeIF from        Leishmania spp., profiling-like protein from Toxoplasma gondii,        Respiratory Syncytial Virus fusion glycoprotein (F-protein),        envelope protein from MMT virus, mouse leukemia virus protein,        Hemagglutinin protein of wild-type measles virus, fungal        immunomodulatory protein (FIP; LZ-8); and Keyhole limpet        hemocyanin (KLH), OspA.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic antibody selected from antibodies used for the        treatment of cancer or tumour diseases, preferably        131I-tositumomab,3F8, 8H9, Abagovomab, Adecatumumab, Afutuzumab,        Alacizumab pegol, Alemtuzumab, Amatuximab, AME-133v, AMG 102,        Anatumomab mafenatox, Apolizumab, Bavituximab, Bectumomab,        Belimumab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab,        Brentuximab vedotin, Cantuzumab, Cantuzumab mertansine,        Cantuzumab ravtansine, Capromab pendetide, Carlumab,        Catumaxomab, Cetuximab, Citatuzumab bogatox, Cixutumumab,        Clivatuzumab tetraxetan, CNTO 328, CNTO95, Conatumumab,        Dacetuzumab, Dalotuzumab, Denosumab, Detumomab, Drozitumab,        Ecromeximab, Edrecolomab, Elotuzumab, Elsilimomab, Enavatuzumab,        Ensituximab, Epratuzumab, Ertumaxomab, Ertumaxomab,        Etaracizumab, Farletuzumab, FBTA05, Ficlatuzumab, Figitumumab,        Flanvotumab, Galiximab, Galiximab, Ganitumab, GC1008,        Gemtuzumab, Gemtuzumab ozogamicin, Girentuximab, Glembatumumab        vedotin, GS6624, HuC242-DM4, HuHMFG1, HuN901-DM1, Ibritumomab,        Icrucumab, ID09C3, Indatuximab ravtansine, Inotuzumab        ozogamicin, Intetumumab, Ipilimumab, Iratumumab, Labetuzumab,        Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab,        Lumiliximab, Mapatumumab, Matuzumab, MDX-060, MEDI 522,        Mitumomab, Mogamulizumab, MORab-003, MORab-009, Moxetumomab        pasudotox, MT103, Nacolomab tafenatox, Naptumomab estafenatox,        Narnatumab, Necitumumab, Nimotuzumab, Nimotuzumab, Olaratumab,        Onartuzumab, Oportuzumab monatox, Oregovomab, Oregovomab, PAM4,        Panitumumab, Patritumab, Pemtumomab, Pertuzumab, Pritumumab,        Racotumomab, Radretumab, Ramucirumab, Rilotumumab, Rituximab,        Robatumumab, Samalizumab, SGN-30, SGN-40, Sibrotuzumab,        Siltuximab,

Tabalumab, Tacatuzumab tetraxetan, Taplitumomab paptox, Tenatumomab,Teprotumumab, TGN1412, Ticilimumab, Tigatuzumab, TNX-650, Tositumomab,Trastuzumab, TRB S07, Tremelimumab, TRU-016, TRU-016, Tucotuzumabcelmoleukin, Ublituximab, Urelumab, Veltuzumab, Veltuzumab (IMMU-106),Volociximab, Votumumab, WX-G250, Zalutumumab, Zanolimumab, Efalizumab,Epratuzumab, Etrolizumab, Fontolizumab, Ixekizumab, Mepolizumab,Milatuzumab, Pooled immunoglobulins, Priliximab, Rituximab,Rontalizumab, Ruplizumab, Sarilumab, Vedolizumab, Visilizumab,Reslizumab, Adalimumab, Aselizumab, Atinumab, Atlizumab, Bertilimumab,Besilesomab, BMS-945429, Briakinumab, Brodalumab, Canakinumab,Canakinumab, Certolizumab pegol, Erlizumab, Fezakinumab, Golimumab,Gomiliximab, Infliximab, Mavrilimumab, Natalizumab, Ocrelizumab,Odulimomab, Ofatumumab, Ozoralizumab, Pexelizumab, Rovelizumab, SBI-087,SBI-087, Secukinumab, Sirukumab, Talizumab, Tocilizumab, Toralizumab,TRU-015, TRU-016, Ustekinumab, Ustekinumab, Vepalimomab, Zolimomabaritox, Sifalimumab, Lumiliximab, and Rho(D) Immune Globulin,Afelimomab, CR6261, Edobacomab, Efungumab, Exbivirumab, Felvizumab,Foravirumab, Ibalizumab, Libivirumab, Motavizumab, Nebacumab, Tuvirumab,Urtoxazumab, Bavituximab, Pagibaximab, Palivizumab, Panobacumab, PRO140, Rafivirumab, Raxibacumab, Regavirumab, Sevirumab, Suvizumab,Tefibazumab, Abciximab, Atorolimumab, Eculizumab, Mepolizumab,Milatuzumab, Antithymocyte globulin, Basiliximab, Cedelizumab,Daclizumab, Gavilimomab, Inolimomab, Muromonab-CD3, Muromonab-CD3,Odulimomab, Siplizumab, Gevokizumab, Otelixizumab, Teplizumab,Bapineuzumab, Crenezumab, Gantenerumab, Ponezumab, R1450, Solanezumab,Benralizumab, Enokizumab, Keliximab, Lebrikizumab, Omalizumab, Oxelumab,Pascolizumab, Tralokinumab, Blosozumab, CaroRx, Fresolimumab,Fulranumab, Romosozumab, Stamulumab, Tanezumab, and Ranibizumab.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;        wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein which is a protein hormone.

The invention further provides the use of such a nucleic acid sequencein gene therapy as defined herein. The invention further provides a kitor kit of parts comprising such a nucleic acid sequence. Further, theinvention provides a pharmaceutical composition comprising such anucleic acid sequence. Further, the invention provides a method forincreasing the expression of an encoded peptide or protein comprisingthe steps of providing such a nucleic acid sequence or a compositioncontaining such a nucleic acid sequence and applying or administeringthe nucleic acid sequence or othe composition to a cell-free expressionsystem, a cell, a tissue or an organism.

Preferably the invention provides nucleic acid sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;        wherein said peptide or protein comprises a therapeutic protein        or a fragment, variant or derivative thereof, preferably a        therapeutic protein which is a protein hormone.    -   The invention further provides the use of such a nucleic acid        sequence in gene therapy as defined herein. The invention        further provides a kit or kit of parts comprising such a nucleic        acid sequence. Further, the invention provides a pharmaceutical        composition comprising such a nucleic acid sequence. Further,        the invention provides a method for increasing the expression of        an encoded peptide or protein comprising the steps of providing        such a nucleic acid sequence or a composition containing such a        nucleic acid sequence and applying or administering the nucleic        acid sequence or othe composition to a cell-free expression        system, a cell, a tissue or an organism.

In the present invention, if not otherwise indicated, different featuresof alternatives and embodiments may be combined with each other.Furthermore, the term “comprising” shall not be construed as meaning“consisting of”, if not specifically mentioned. However, in the contextof the present invention, term “comprising” may be substituted with theterm “consisting of”, where applicable.

BRIEF DESCRIPTION OF THE FIGURES

The following Figures are intended to illustrate the invention furtherand shall not be construed to limit the present invention thereto.

FIG. 1: shows the histone stem-loop consensus sequence generated frommetazoan and protozoan stem loop sequences (as reported by Dávila López,M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308). 4001 histone stem-loop sequences from metazoaand protozoa were aligned and the quantity of the occurring nucleotidesis indicated for every position in the stem-loop sequence. The generatedconsensus sequence representing all nucleotides present in the sequencesanalyzed is given using the single-letter nucleotide code. In additionto the consensus sequence, sequences are shown representing at least99%, 95% and 90% of the nucleotides present in the sequences analyzed.

FIG. 2: shows the histone stem-loop consensus sequence generated fromprotozoan stem loop sequences (as reported by Dávila López, M., &Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308). 131 histone stem-loop sequences from protozoawere aligned and the quantity of the occurring nucleotides is indicatedfor every position in the stem-loop sequence. The generated consensussequence representing all nucleotides present in the sequences analyzedis given using the single-letter nucleotide code. In addition to theconsensus sequence, sequences are shown representing at least 99%, 95%and 90% of the nucleotides present in the sequences analyzed.

FIG. 3: shows the histone stem-loop consensus sequence generated frommetazoan stem loop sequences (as reported by Dávila López, M., &Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308). 3870 histone stem-loop sequences from metazoawere aligned and the quantity of the occurring nucleotides is indicatedfor every position in the stem-loop sequence. The generated consensussequence representing all nucleotides present in the sequences analyzedis given using the single-letter nucleotide code. In addition to theconsensus sequence, sequences are shown representing at least 99%, 95%and 90% of the nucleotides present in the sequences analyzed.

FIG. 4: shows the histone stem-loop consensus sequence generated fromvertebrate stem loop sequences (as reported by Dávila López, M., &Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308). 1333 histone stem- loop sequences fromvertebrates were aligned and the quantity of the occurring nucleotidesis indicated for every position in the stem-loop sequence. The generatedconsensus sequence representing all nucleotides present in the sequencesanalyzed is given using the single-letter nucleotide code. In additionto the consensus sequence, sequences are shown representing at least99%, 95% and 90% of the nucleotides present in the sequences analyzed.

FIG. 5: shows the histone stem-loop consensus sequence generated fromhuman (Homo sapiens) stem loop sequences (as reported by Dávila López,M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308). 84 histone stem-loop sequences from humans werealigned and the quantity of the occurring nucleotides is indicated forevery position in the stem-loop sequence. The generated consensussequence representing all nucleotides present in the sequences analyzedis given using the single-letter nucleotide code. In addition to theconsensus sequence, sequences are shown representing at least 99%, 95%and 90% of the nucleotides present in the sequences analyzed.

FIGS. 6 to 19: show mRNAs from in vitro transcription.

-   -   Given are the designation and the sequence of mRNAs obtained by        in vitro transcription. The following abbreviations are used:    -   ppLuc (GC): GC-enriched mRNA sequence coding for Photinus        pyralis luciferase    -   ag: 3′ untranslated region (UTR) of the alpha globin gene    -   A64: poly(A)-sequence with 64 adenylates    -   A120: poly(A)-sequence with 120 adenylates    -   histoneSL: histone stem-loop    -   aCPSL: stem loop which has been selected from a library for its        specific binding of the αCP-2KL protein    -   PolioCL: 5′ clover leaf from Polio virus genomic RNA    -   G30: poly(G) sequence with 30 guanylates    -   U30: poly(U) sequence with 30 uridylates    -   SL: unspecific/artificial stem-loop    -   N32: unspecific sequence of 32 nucleotides    -   MmEPO (GC): GC-enriched mRNA sequence coding for murine EPO    -   Within the sequences, the following elements are highlighted:        coding region (ORF) (capital letters), ag (bold), histoneSL        (underlined), further distinct sequences tested (italic).

FIG. 6: shows the mRNA sequence of ppLuc(GC)-ag (SEQ ID NO: 43).

-   -   By linearization of the original vector at the restriction site        immediately following the alpha-globin 3′-UTR (ag), mRNA is        obtained lacking a poly(A) sequence.

FIG. 7: shows the mRNA sequence of ppLuc(GC)-ag-A64 (SEQ ID NO: 44).

-   -   By linearization of the original vector at the restriction site        immediately following the A64 poly(A)-sequence, mRNA is obtained        ending with an A64 poly(A) sequence.

FIG. 8: shows the mRNA sequence of ppLuc(GC)-ag-histoneSL (SEQ ID NO:45).

-   -   The A64 poly(A) sequence was replaced by a histoneSL. The        histone stem-loop sequence used in the examples was obtained        from Cakmakci et al. (2008). Molecular and Cellular Biology,        28(3), 1182-1194.

FIG. 9: shows the mRNA sequence of ppLuc(GC)-ag-A64-histoneSL (SEQ IDNO: 46).

-   -   The histoneSL was appended 3′ of A64 poly(A).

FIG. 10: shows the mRNA sequence of ppLuc(GC)-ag-A120 (SEQ ID NO: 47).

-   -   The A64 poly(A) sequence was replaced by an A120 poly(A)        sequence.

FIG. 11: shows the mRNA sequence of ppLuc(GC)-ag-A64-ag (SEQ ID NO: 48).A second alpha-globin 3′ -UTR was appended 3′ of A64 poly(A).

FIG. 12: shows the mRNA sequence of ppLuc(GC)-ag-A64-αCPSL (SEQ ID NO:49). A stem loop was appended 3′ of A64 poly(A). The stem loop has beenselected from a library for its specific binding of the αCP-2KL protein(Thisted et al., (2001), The Journal of Biological Chemistry, 276(20),17484-17496). αCP-2KL is an isoform of αCP-2, the most stronglyexpressed αCP protein (alpha-globin mRNA poly(C) binding protein)(Makeyev et al., (2000), Genomics, 67(3), 301-316), a group of RNAbinding proteins, which bind to the alpha-globin 3′-UTR (Chkheidze etal., (1999), Molecular and Cellular Biology, 19(7), 4572-4581).

FIG. 13: shows the mRNA sequence of ppLuc(GC)-ag-A64-PolioCL (SEQ ID NO:50).

-   -   The 5′ clover leaf from Polio virus genomic RNA was appended 3′        of A64 poly(A).

FIG. 14: shows the mRNA sequence of ppLuc(GC)-ag-A64-G30 (SEQ ID NO: 51)A stretch of 30 guanylates was appended 3′ of A64 poly(A).

FIG. 15: shows the mRNA sequence of ppLuc(GC)-ag-A64-U30 (SEQ ID NO: 52)A stretch of 30 uridylates was appended 3′ of A64 poly(A).

FIG. 16: shows the mRNA sequence of ppLuc(GC)-ag-A64-SL (SEQ ID NO: 53)A stem loop was appended 3′ of A64 poly(A). The upper part of the stemand the loop were taken from (Babendure et al., (2006), RNA (New York,N.Y.), 12(5), 851-861). The stem loop consists of a 17 base pair long,CG-rich stem and a 6 base long loop.

FIG. 17: shows ppLuc(GC)-ag-A64-N32 (SEQ ID NO: 54)

-   -   By linearization of the original vector at an alternative        restriction site, mRNA is obtained with 32 additional        nucleotides following poly(A).

FIG. 18: shows the mRNA sequence of MmEPO (GC)-ag-A64-C30 (SEQ ID NO:55)

FIG. 19: shows the mRNA sequence of MmEPO (GC)-ag-A64-C30-histoneSL (SEQID NO: 56)

FIG. 20: shows that the combination of poly(A) and histoneSL increasesprotein expression from mRNA in a synergistic manner.

-   -   The effect of poly(A) sequence, histoneSL, and the combination        of poly(A) and histoneSL on luciferase expression from mRNA was        examined. Therefore different mRNAs were electroporated into        HeLa cells. Luciferase levels were measured at 6, 24, and 48        hours after transfection. Little luciferase is expressed from        mRNA having neither poly(A) sequence nor histoneSL. Both a        poly(A) sequence or the histoneSL increase the luciferase level.        Strikingly however, the combination of poly(A) and histoneSL        further strongly increases the luciferase level, manifold above        the level observed with either of the individual elements, thus        acting synergistically. Data are graphed as mean RLU±SD        (relative light units±standard deviation) for triplicate        transfections. Specific RLU are summarized in Example 10.2.

FIG. 21: shows that the combination of poly(A) and histoneSL increasesprotein expression from mRNA irrespective of their order.

-   -   The effect of poly(A) sequence, histoneSL, the combination of        poly(A) and histoneSL, and their order on luciferase expression        from mRNA was examined. Therefore different mRNAs were        lipofected into HeLa cells. Luciferase levels were measured at        6, 24, and 48 hours after the start of transfection. Both an A64        poly(A) sequence or the histoneSL give rise to comparable        luciferase levels. Increasing the length of the poly(A) sequence        from A64 to A120 or to A300 increases the luciferase level        moderately. In contrast, the combination of poly(A) and        histoneSL increases the luciferase level much further than        lengthening of the poly(A) sequence. The combination of poly(A)        and histoneSL acts synergistically as it increases the        luciferase level manifold above the level observed with either        of the individual elements. The synergistic effect of the        combination of poly(A) and histoneSL is seen irrespective of the        order of poly(A) and histoneSL and irrespective of the length of        poly(A) with A64-histoneSL or histoneSL-A250 mRNA. Data are        graphed as mean RLU±SD for triplicate transfections. Specific        RLU are summarized in Example 10.3.

FIG. 22: shows that the rise in protein expression by the combination ofpoly(A) and histoneSL is specific.

-   -   The effect of combining poly(A) and histoneSL or poly(A) and        alternative sequences on luciferase expression from mRNA was        examined. Therefore different mRNAs were electroporated into        HeLa cells. Luciferase levels were measured at 6, 24, and 48        hours after transfection. Both a poly(A) sequence or the        histoneSL give rise to comparable luciferase levels. The        combination of poly(A) and histoneSL strongly increases the        luciferase level, manifold above the level observed with either        of the individual elements, thus acting synergistically. In        contrast, combining poly(A) with any of the other sequences is        without effect on the luciferase level compared to mRNA        containing only a poly(A) sequence. Thus, the combination of        poly(A) and histoneSL acts specifically and synergistically.        Data are graphed as mean RLU±SD for triplicate transfections.        Specific RLU are summarized in Example 10.4.

FIG. 23: shows that the combination of poly(A) and histoneSL increasesprotein expression from mRNA in a synergistic manner in vivo.

-   -   The effect of poly(A) sequence, histoneSL, and the combination        of poly(A) and histoneSL on luciferase expression from mRNA in        vivo was examined. Therefore different mRNAs were injected        intradermally into mice. Mice were sacrificed 16 hours after        injection and Luciferase levels at the injection sites were        measured. Luciferase is expressed from mRNA having either a        histoneSL or a poly(A) sequence. Strikingly however, the        combination of poly(A) and histoneSL strongly increases the        luciferase level, manifold above the level observed with either        of the individual elements, thus acting synergistically. Data        are graphed as mean RLU±SEM (relative light units±standard error        of mean). Specific RLU are summarized in Example 10.5.

FIG. 24: shows the mRNA sequence of Trastuzumab (GC)-ag-A64-C30 (SEQ IDNO: 57). This sequence encodes the antibody Trastuzumab (Herceptin)comprising the light and heavy chains as described in WO2008/083949.

FIG. 25: shows the mRNA sequence of Trastuzumab(GC)-ag-A64-C30-histoneSL (SEQ ID NO: 58). This sequence encodes theantibody Trastuzumab (Herceptin) comprising the light and heavy chainsas described in WO2008/083949.

EXAMPLES

The following Examples are intended to illustrate the invention furtherand shall not be construed to limit the present invention thereto.

1. Generation of Histone-Stem-Loop Consensus Sequences

Prior to the experiments, histone stem-loop consensus sequences weredetermined on the basis of metazoan and protozoan histone stem-loopsequences. Sequences were taken from the supplement provided by Lopez etal. (Dávila López, M., & Samuelsson, T. (2008), RNA (New York, N.Y.),14(1), 1-10. doi:10.1261/rna.782308), who identified a large number ofnatural histone stem-loop sequences by searching genomic sequences andexpressed sequence tags. First, all sequences from metazoa and protozoa(4001 sequences), or all sequences from protozoa (131 sequences) oralternatively from metazoa (3870 sequences), or from vertebrates (1333sequences) or from humans (84 sequences) were grouped and aligned. Then,the quantity of the occurring nucleotides was determined for everyposition. Based on the tables thus obtained, consensus sequences for the5 different groups of sequences were generated representing allnucleotides present in the sequences analyzed. In addition, morerestrictive consensus sequences were also obtained, increasinglyemphasizing conserved nucleotides

2. Preparation of DNA-Templates

A vector for in vitro transcription was constructed containing a T7promoter followed by a GC-enriched sequence coding for Photinus pyralisluciferase (ppLuc(GC)), the center part of the 3′ untranslated region(UTR) of alpha-globin (ag), and a poly(A) sequence. The poly(A) sequencewas immediately followed by a restriction site used for linearization ofthe vector before in vitro transcription in order to obtain mRNA endingin an A64 poly(A) sequence. mRNA obtained from this vector accordinglyby in vitro transcription is designated as “ppLuc(GC)-ag-A64”.

Linearization of this vector at alternative restriction sites before invitro transcription allowed to obtain mRNA either extended by additionalnucleotides 3′ of A64 or lacking A64. In addition, the original vectorwas modified to include alternative sequences. In summary, the followingmRNAs were obtained from these vectors by in vitro transcription (mRNAsequences are given in FIGS. 6 to 17):

(SEQ ID NO: 43) ppLuc(GC)-ag (SEQ ID NO: 44) ppLuc(GC)-ag-A64(SEQ ID NO: 45) ppLuc(GC)-ag-histoneSL (SEQ ID NO: 46)ppLuc(GC)-ag-A64-histoneSL (SEQ ID NO: 47) ppLuc(GC)-ag-A120(SEQ ID NO: 48) ppLuc(GC)-ag-A64-ag (SEQ ID NO: 49)ppLuc(GC)-ag-A64-aCPSL (SEQ ID NO: 50) ppLuc(GC)-ag-A64-PolioCL(SEQ ID NO: 51) ppLuc(GC)-ag-A64-G30 (SEQ ID NO: 52)ppLuc(GC)-ag-A64-U30 (SEQ ID NO: 53) ppLuc(GC)-ag-A64-SL (SEQ ID NO: 54)ppLuc(GC)-ag-A64-N32

Furthermore DNA plasmid sequences coding for the therapeutic protein EPOwas prepared accordingly as described above.

In summary, the following mRNAs were obtained from these vectors by invitro transcription (mRNA sequences are given in FIGS. 18 to 19):

(SEQ ID NO: 55) MmEPO (GC)-ag-A64-C30  (SEQ ID NO: 56)MmEPO (GC)-ag-A64-C30-histoneSL 

Furthermore DNA plasmid sequences coding for the antibody Trastuzumabcan be prepared accordingly as described above.

The following mRNAs are obtained from these vectors by in vitrotranscription (mRNA sequences are given in FIGS. 24 and 25):

(SEQ ID NO: 57) Trastuzumab (GC)-ag-A64-C30  (SEQ ID NO: 58)Trastuzumab (GC)-ag-A64-C30-histoneSL 3. In vitro Transcription

The DNA-template according to Example 2 was linearized and transcribedin vitro using T7-Polymerase. The DNA-template was then digested byDNase-treatment. All mRNA-transcripts contained a 5′-CAP structureobtained by adding an excess ofN7-Methyl-Guanosine-5′-Triphosphate-5′-Guanosine to the transcriptionreaction. mRNA thus obtained was purified and resuspended in water.

4. Enzymatic Adenylation of mRNA

Two mRNAs were enzymatically adenylated:

ppLuc(GC)-ag-A64 and ppLuc(GC)-ag-histoneSL.

To this end, RNA was incubated with E. coli Poly(A)-polymerase and ATP(Poly(A) Polymerase Tailing Kit, Epicentre, Madison, USA) following themanufacturer's instructions. mRNA with extended poly(A) sequence waspurified and resuspended in water. The length of the poly(A) sequencewas determined via agarose gel electrophoresis. Starting mRNAs wereextended by approximately 250 adenylates, the mRNAs obtained aredesignated as

ppLuc(GC)-ag-A300 and ppLuc(GC)-ag-histoneSL-A250, respectively.

5. Luciferase Expression by mRNA Electroporation

HeLa cells were trypsinized and washed in OPTI-MEM®. 1×10⁵ cells in 200μl of opti-MEM each were electroporated with 0.5 μg of ppLuc-encodingmRNA. As a control, mRNA not coding for ppLuc was electroporatedseparately. Electroporated cells were seeded in 24-well plates in 1 mlof RPMI 1640 medium. 6, 24, or 48 hours after transfection, medium wasaspirated and cells were lysed in 200 μl of lysis buffer (25 mM Tris, pH7.5 (HCl), 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM DTT, 1 mMPMSF). Lysates were stored at −20° C. until ppLuc activity was measured.

6. Luciferase Expression by mRNA Lipofection

HeLa cells were seeded in 96 well plates at a density of 2×10⁴ cells perwell. The following day, cells were washed in OPTI-MEM® and thentransfected with 0.25 μg of Lipofectin-complexed ppLuc-encoding mRNA in150 μl of OPTI-MEM®. As a control, mRNA not coding for ppLuc waslipofected separately. In some wells, OPTI-MEM® was aspirated and cellswere lysed in 200 μl of lysis buffer 6 hours after the start oftransfection. In the remaining wells, OPTI-MEM® was exchanged for RPMI1640 medium at that time. In these wells, medium was aspirated and cellswere lysed in 200 μl of lysis buffer 24 or 48 hours after the start oftransfection. Lysates were stored at −20° C. until ppLuc activity wasmeasured.

7. Luciferase Measurement

ppLuc activity was measured as relative light units (RLU) in a BioTekSYNERGY™HT plate reader at 5 seconds measuring time using 50 μl oflysate and 200 μl of luciferin buffer (25 mM Glycylglycin, pH 7.8(NaOH), 15 mM MgSO₄, 2 mM ATP, 75 μM luciferin). Specific RLU werecalculated by subtracting RLU of the control RNA from total RLU.

8. Luciferase Expression by Intradermal mRNA Injection (LuciferaseExpression in vivo)

Mice were anaesthetized with a mixture of ROMPUN™ and KETAVET™. EachppLuc-encoding mRNA was injected intradermally (0.5 μg of mRNA in 50 μlper injection). As a control, mRNA not coding for ppLuc was injectedseparately. 16 hours after injection, mice were sacrificed and tissuecollected. Tissue samples were flash frozen in liquid nitrogen and lysedin a tissue lyser (Qiagen) in 800 μl of lysis buffer (25 mM Tris, pH 7.5(HCl), 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM DTT, 1 mM PMSF).Subsequently samples were centrifuged at 13500 rpm at 4° C. for 10minutes. Lysates were stored at −80° C. until ppLuc activity wasmeasured (see 7. luciferase measurement).

9. MmEPO Expression in HeLa Cells

HeLa cells are trypsinized and washed in OPTI-MEM®. 1×10⁵ cells in 20082 l of OPTI-MEM® each are electroporated with 0.5 μg of MmEPO-encodingmRNA. As a control, irrelevant mRNA is electroporated separately.Electroporated cells are seeded in 24-well plates in 1 ml of RPMI 1640medium. 6, 24, or 48 hours after transfection, supernatants are takenand harvested from the cells. The content of EPO in the supernatants ismeasured with the Mouse/Rat Erythropoietin QUANTIKINE® ELISA Kit (R&DSystems) according to the manufacturer's instructions.

10. Results

10.1 Histone Stem-Loop Sequences:

In order to characterize histone stem-loop sequences, sequences frommetazoa and protozoa (4001 sequences), or from protozoa (131 sequences)or alternatively from metazoa (3870 sequences), or from vertebrates(1333 sequences) or from humans (84 sequences) were grouped and aligned.Then, the quantity of the occurring nucleotides was determined for everyposition. Based on the tables thus obtained, consensus sequences for the5 different groups of sequences were generated representing allnucleotides present in the sequences analyzed. Within the consensussequence of metazoa and protozoa combined, 3 nucleotides are conserved,a T/U in the loop and a G and a C in the stem, forming a base pair.Structurally, typically a 6 base-pair stem and a loop of 4 nucleotidesis formed. However, deviating structures are common: Of 84 human histonestem-loops, two contain a stem of only 5 nucleotides comprising 4base-pairs and one mismatch. Another human histone stem-loop contains astem of only 5 base-pairs. Four more human histone stem-loops contain a6 nucleotide long stem, but include one mismatch at three differentpositions, respectively. Furthermore, four human histone stem-loopscontain one wobble base-pair at two different positions, respectively.Concerning the loop, a length of 4 nucleotides seems not to be strictlyrequired, as a loop of 5 nucleotides has been identified in D.discoideum.

In addition to the consensus sequences representing all nucleotidespresent in the sequences analyzed, more restrictive consensus sequenceswere also obtained, increasingly emphasizing conserved nucleotides. Insummary, the following sequences were obtained:

(Cons): represents all nucleotides present

(99%): represents at least 99% of all nucleotides present

(95%): represents at least 95% of all nucleotides present

(90%): represents at least 90% of all nucleotides present

The results of the analysis of histone stem-loop sequences aresummarized in the following Tables 1 to 5 (see also FIGS. 1 to 5):

TABLE 1 Metzoan and protozoan histone stem-loop consensus sequence:(based on an alignment of 4001 metazoan and protozoan histone stem-loopsequences) (see also FIG. 1) < < < < < < • • # A 2224 1586 3075 28721284 184 0 13 12 9 1 47 59 # T 172 188 47 205 19 6 0 569 1620 199 39473830 3704 # C 1557 2211 875 918 2675 270 0 3394 2342 3783 51 119 227 # G25 16 4 6 23 3541 4001 25 27 10 2 5 11 Cons N* N* N N N N G N N N N N N99% H* H* H H V V G Y Y Y Y H H 95% M* H* M H M S G Y Y Y T T Y 90% M*M* M M M S G Y Y C T T T • • > > > > > > # A 0 675 3818 195 1596 523 014 3727 61 771 2012 2499 # T 4001 182 1 21 15 11 0 179 8 64 557 201 690# C 0 3140 7 50 31 16 4001 3543 154 3870 2636 1744 674 # G 0 4 175 37352359 3451 0 265 112 4 37 43 138 Cons T N N N N N C N N N N* N* N* 99% TH R V V R C B V H H* N* N* 95% T M A R R R C S M C H* H* H* 90% T M A GR R C S A C H* M* H*

TABLE 2 Protozoan histone stem-loop consensus sequence: (based on analignment of 131 protozoan histone stem-loop sequences) (see also FIG.2) < < < < < < • • • • > > > > > > # A 52 32 71 82 76 13 0 12 12 9 1 463 0 75 82 53 79 20 0 4 94 17 35 74 56 # T 20 32 37 21 8 3 0 21 85 58 8670 65 131 28 1 17 13 10 0 15 7 31 32 20 28 # C 45 59 20 25 38 0 0 86 854 42 13 58 0 27 2 6 31 10 131 112 5 82 58 30 40 # G 14 8 3 3 9 115 13112 26 10 2 2 5 0 1 46 55 8 91 0 0 25 1 6 7 7 Cons N* N* N N N D G N N NN N N T N N N N N C H N N N* N* N* 99% N* N* N N N D G N N N B N N T H VN N N C H N H N* N* N* 95% N* N* H H N R G N N N Y H B T H R D N N C Y DH H* N* N* 90% N* H* H H V R G N D B Y H Y T H R D H N C Y R H H* H* H*

TABLE 3 Metazoan histone stem-loop consensus sequence: (based on analignment of 3870 (including 1333 vertebrate sequences) metazoan histonestem-loop sequences) (see also FIG. 3) < < < < < < • • # A 2172 15543004 2790 1208 171 0 1 0 0 0 1 56 # T 152 156 10 184 11 3 0 548 1535 1413861 3760 3639 # C 1512 2152 855 893 2637 270 0 3308 2334 3729 9 106 169# G 11 8 1 3 14 3426 3870 13 1 0 0 3 6 Cons N* N* N N N N G N B Y Y N N99% H* H* M H M V G Y Y Y T Y H 95% M* M* M M M S G Y Y C T T Y 90% M*M* M M M S G Y Y C T T T • • > > > > > > # A 0 600 3736 142 1517 503 010 3633 44 736 1938 2443 # T 3870 154 0 4 2 1 0 164 1 33 525 181 662 # C0 3113 5 44 0 6 3870 3431 149 3788 2578 1714 634 # G 0 3 129 3680 23513360 0 265 87 3 31 36 131 Cons T N V N D N C N N N N* N* N* 99% T H R VR R C B V M H* H* N* 95% T M A G R R C S M C H* H* H* 90% T M A G R R CS A C H* M* H*

TABLE 4 Vertebrate histone stem-loop consensus sequence: (based on analignment of 1333 vertebrate histone stem-loop sequences) (see also FIG.4) < < < < < < • • # A 661 146 1315 1323 920 8 0 1 0 0 0 1 4 # T 63 1212 2 6 2 0 39 1217 2 1331 1329 1207 # C 601 1062 16 6 403 1 0 1293 1161331 2 0 121 # G 8 4 0 2 4 1322 1333 0 0 0 0 3 1 Cons N* N* H N N N G HY Y Y D N 99% H* H* M A M G G Y Y C T T Y 95% H* H* A A M G G C Y C T TY 90% M* M* A A M G G C T C T T T • • > > > > > > # A 0 441 1333 0 119921 0 1 1126 26 81 380 960 # T 1333 30 0 1 0 1 0 2 1 22 91 91 12 # C 0862 0 2 0 0 1333 1328 128 1284 1143 834 361 # G 0 0 0 1330 134 1311 0 278 1 18 28 0 Cons T H A B R D C N N N N* N* H* 99% T H A G R R C C V HN* N* M* 95% T M A G R G C C V C H* H* M* 90% T M A G R G C C M C Y* M*M*

TABLE 5 Homo sapiens histone stem-loop consensus sequence: (based on analignment of 84 human histone stem-loop sequences) (see also FIG. 5) < << < < < • • • • > > > > > > # A 10 17 84 84 76 1 0 1 0 0 0 1 0 0 12 84 065 3 0 0 69 5 0 10 64 # T 8 6 0 0 2 2 0 1 67 0 84 80 81 84 5 0 0 0 0 0 00 4 25 24 3 # C 62 61 0 0 6 0 0 82 17 84 0 0 3 0 67 0 1 0 0 84 84 5 7557 44 17 # G 4 0 0 0 0 81 84 0 0 0 0 3 0 0 0 0 83 19 81 0 0 10 0 2 6 0Cons N* H* A A H D G H Y C T D Y T H A S R R C C V H B* N* H* 99% N* H*A A H D G H Y C T D Y T H A S R R C C V H B* N* H* 95% H* H* A A M G G CY C T T T T H A G R G C C V M Y* N* M* 90% H* M* A A A G G C Y C T T T TM A G R G C C R M Y* H* M*

Wherein the used abbreviations were defined as followed:

abbreviation Nucleotide bases remark G G Guanine A A Adenine T T ThymineU U Uracile C C Cytosine R G or A Purine Y T/U or C Pyrimidine M A or CAmino K G or T/U Keto S G or C Strong (3H bonds) W A or T/U Weak (2Hbonds) H A or C or T/U Not G B G or T/U or C Not A V G or C or A Not T/UD G or A or T/U Not C N G or C or T/U or A Any base * present or notBase may be present or not10.2 The Combination of poly(A) and HistoneSL Increases ProteinExpression from mRNA in a Synergistic Manner.

To investigate the effect of the combination of poly(A) and histoneSL onprotein expression from mRNA, mRNAs with different sequences 3′ of thealpha-globin 3′-UTR were synthesized: mRNAs either ended just 3′ of the3′-UTR, thus lacking both poly(A) sequence and histoneSL, or containedeither an A64 poly(A) sequence or a histoneSL instead, or both A64poly(A) and histoneSL 3′ of the 3′-UTR. Luciferase-encoding mRNAs orcontrol mRNA were electroporated into HeLa cells. Luciferase levels weremeasured at 6, 24, and 48 hours after transfection (see following Table6 and FIG. 20).

TABLE 6 RLU at RLU at RLU at mRNA 6 hours 24 hours 48 hoursppLuc(GC)-ag-A64-histoneSL 466553 375169 70735 ppLuc(GC)-ag-histoneSL50947 3022 84 ppLuc(GC)-ag-A64 10471 19529 4364 ppLuc(GC)-ag 997 217 42

Little luciferase was expressed from mRNA having neither poly(A)sequence nor histoneSL. Both a poly(A) sequence or the histoneSLincreased the luciferase level to a similar extent. Either mRNA gaverise to a luciferase level much higher than did mRNA lacking bothpoly(A) and histoneSL. Strikingly however, the combination of poly(A)and histoneSL further strongly increased the luciferase level, manifoldabove the level observed with either of the individual elements. Themagnitude of the rise in luciferase level due to combining poly(A) andhistoneSL in the same mRNA demonstrates that they are actingsynergistically.

The synergy between poly(A) and histoneSL was quantified by dividing thesignal from poly(A)-histoneSL mRNA (+/+) by the sum of the signals fromhistoneSL mRNA (−/+) plus poly(A) mRNA (+/−) (see following Table 7).

TABLE 7 RLU at RLU at RLU at A64 histoneSL 6 hours 24 hours 48 hours + +466553 375169 70735 − + 50947 3022 84 + − 10471 19529 4364 Synergy 7.616.6 15.9

The factor thus calculated specifies how much higher the luciferaselevel from mRNA combining poly(A) and histoneSL is than would beexpected if the effects of poly(A) and histoneSL were purely additive.The luciferase level from mRNA combining poly(A) and histoneSL was up to16.6 times higher than if their effects were purely additive. Thisresult confirms that the combination of poly(A) and histoneSL effects amarkedly synergistic increase in protein expression.

10.3 The Combination of Poly(A) and HistoneSL Increases ProteinExpression from mRNA Irrespective of their Order.

The effect of the combination of poly(A) and histoneSL might depend onthe length of the poly(A) sequence and the order of poly(A) andhistoneSL. Thus, mRNAs with increasing poly(A) sequence length and mRNAwith poly(A) and histoneSL in reversed order were synthesized: Two mRNAscontained 3′ of the 3′-UTR either an A120 or an A300 poly(A) sequence.One further mRNA contained 3′ of the 3′-UTR first a histoneSL followedby an A250 poly(A) sequence. Luciferase-encoding mRNAs or control mRNAwere lipofected into HeLa cells. Luciferase levels were measured at 6,24, and 48 hours after the start of transfection (see following Table 8and FIG. 21).

TABLE 8 RLU at RLU at RLU at mRNA 6 hours 24 hours 48 hoursppLuc(GC)-ag-histoneSL-A250 98472 734222 146479ppLuc(GC)-ag-A64-histoneSL 123674 317343 89579 ppLuc(GC)-ag-histoneSL7291 4565 916 ppLuc(GC)-ag-A300 4357 38560 11829 ppLuc(GC)-ag-A120 437145929 10142 ppLuc(GC)-ag-A64 1928 26781 537

Both an A64 poly(A) sequence or the histoneSL gave rise to comparableluciferase levels. In agreement with the previous experiment did thecombination of A64 and histoneSL strongly increase the luciferase level,manifold above the level observed with either of the individualelements. The magnitude of the rise in luciferase level due to combiningpoly(A) and histoneSL in the same mRNA demonstrates that they are actingsynergistically. The synergy between A64 and histoneSL was quantified asbefore based on the luciferase levels of A64-histoneSL, A64, andhistoneSL mRNA (see following Table 9). The luciferase level from mRNAcombining A64 and histoneSL was up to 61.7 times higher than if theeffects of poly(A) and histoneSL were purely additive.

TABLE 9 RLU at RLU at RLU at A64 histoneSL 6 hours 24 hours 48 hours + +123674 317343 89579 − + 7291 4565 916 + − 1928 26781 537 Synergy 13.410.1 61.7

In contrast, increasing the length of the poly(A) sequence from A64 toA120 or to A300 increased the luciferase level only moderately (seeTable 8 and FIG. 19). mRNA with the longest poly(A) sequence, A300, wasalso compared to mRNA in which a poly(A) sequence of similar length wascombined with the histoneSL, histoneSL-A250. In addition to having along poly(A) sequence, the order of histoneSL and poly(A) is reversed inthis mRNA relative to A64-histoneSL mRNA. The combination of A250 andhistoneSL strongly increased the luciferase level, manifold above thelevel observed with either histoneSL or A300. Again, the synergy betweenA250 and histoneSL was quantified as before comparing RLU fromhistoneSL-A250 mRNA to RLU from A300 mRNA plus histoneSL mRNA (seefollowing Table 10). The luciferase level from mRNA combining A250 andhistoneSL was up to 17.0 times higher than if the effects of poly(A) andhistoneSL were purely additive.

TABLE 10 RLU at RLU at RLU at histoneSL A250/A300 6 hours 24 hours 48hours + + 98472 734222 146479 + − 7291 4565 916 − + 4357 38560 11829Synergy 8.5 17.0 11.5

In summary, a highly synergistic effect of the combination of histoneSLand poly(A) on protein expression from mRNA has been demonstrated forsubstantially different lengths of poly(A) and irrespective of the orderof poly(A) and histoneSL.

10.4 The Rise in Protein Expression by the Combination of Poly(A) andHistoneSL is Specific

To investigate whether the effect of the combination of poly(A) andhistoneSL on protein expression from mRNA is specific, mRNAs withalternative sequences in combination with poly(A) were synthesized:These mRNAs contained 3′ of A64 one of seven distinct sequences,respectively. Luciferase-encoding mRNAs or control mRNA wereelectroporated into HeLa cells. Luciferase levels were measured at 6,24, and 48 hours after transfection (see following Table 11 and FIG.22).

TABLE 11 RLU at RLU at RLU at mRNA 6 hours 24 hours 48 hoursppLuc(GC)-ag-A64-N32 33501 38979 2641 ppLuc(GC)-ag-A64-SL 28176 20364874 ppLuc(GC)-ag-A64-U30 41632 54676 3408 ppLuc(GC)-ag-A64-G30 4676349210 3382 ppLuc(GC)-ag-A64-PolioCL 46428 26090 1655ppLuc(GC)-ag-A64-aCPSL 34176 53090 3338 ppLuc(GC)-ag-A64-ag 18534 18194989 ppLuc(GC)-ag-A64-histoneSL 282677 437543 69292ppLuc(GC)-ag-histoneSL 27597 3171 0 ppLuc(GC)-ag-A64 14339 48414 9357

Both a poly(A) sequence or the histoneSL gave rise to comparableluciferase levels. Again, the combination of poly(A) and histoneSLstrongly increased the luciferase level, manifold above the levelobserved with either of the individual elements, thus actingsynergistically. In contrast, combining poly(A) with any of thealternative sequences was without effect on the luciferase levelcompared to mRNA containing only a poly(A) sequence. Thus, thecombination of poly(A) and histoneSL increases protein expression frommRNA in a synergistic manner, and this effect is specific.

10.5 The Combination of Poly(A) and HistoneSL Increases ProteinExpression from mRNA in a Synergistic Manner in vivo.

To investigate the effect of the combination of poly(A) and histoneSL onprotein expression from mRNA in vivo, Luciferase-encoding mRNAs withdifferent sequences 3′ of the alpha-globin 3′-UTR or control mRNA wereinjected intradermally into mice: mRNAs contained either an A64 poly(A)sequence or a histoneSL instead, or both A64 poly(A) and histoneSL 3′ ofthe 3′-UTR. Luciferase levels were measured at 16 hours after injection(see following Table 12 and FIG. 23).

TABLE 12 RLU at mRNA 16 hours ppLuc(GC)-ag-A64-histoneSL 38081ppLuc(GC)-ag-histoneSL 137 ppLuc(GC)-ag-A64 4607

Luciferase was expressed from mRNA having either a histoneSL or apoly(A) sequence. Strikingly, however, the combination of poly(A) andhistoneSL further strongly increased the luciferase level, manifoldabove the level observed with either of the individual elements. Themagnitude of the rise in luciferase level due to combining poly(A) andhistoneSL in the same mRNA demonstrates that they are actingsynergistically.

The synergy between poly(A) and histoneSL was quantified by dividing thesignal from poly(A)-histoneSL mRNA (+/+) by the sum of the signals fromhistoneSL mRNA (−/+) plus poly(A) mRNA (+/−) (see following Table 13).

TABLE 13 RLU at A64 histoneSL 16 hours + + 38081 − + 137 + − 4607Synergy 8.0

The factor thus calculated specifies how much higher the luciferaselevel from mRNA combining poly(A) and histoneSL is than would beexpected if the effects of poly(A) and histoneSL were purely additive.The luciferase level from mRNA combining poly(A) and histoneSL was 8times higher than if their effects were purely additive. This resultconfirms that the combination of poly(A) and histoneSL effects amarkedly synergistic increase in protein expression in vivo.

11. Antibody Expression and Characterization

Cell lines

RNA-based expression of humanised antibodies is done in either CHO-K1 orBHK-21 (Syrian hamster kidney, HER2-negative) cells. The tumour cellline BT-474 strongly expresses HER2 and is used to record antibodylevels by FACS analysis. All cell lines except CHO are maintained inRPMI medium supplemented with FCS and glutamine according to thesupplier's information. CHO cells are grown in Ham's F12 supplementedwith 10% FCS. All cell lines can be obtained from the German collectionof cell cultures (DSMZ, Braunschweig, Germany).

Antibody expression

Various amounts of mRNA (G/C enriched as defined by the FIGS. 24 and 25)encoding the humanised antibody Herceptin (Trastuzumab) is transfectedinto either CHO or BHK cells by electroporation (300 V, 450 μF for CHOand 300 V, 150 μF for BHK). After transfection, cells are seeded onto24-well cell culture plates at a density of 200.000 to 400.000 cells perwell. For collection of secreted protein, medium is replaced by 250 μlof fresh medium after cell attachment to the plastic surface. Secretedprotein is collected for 24-96 hours and stored at 4° C. In addition,cells are harvested into 50 μl of phosphate buffered saline (1× PBSbuffer) containing 0.5% BSA and are disrupted by three freeze-thawcycles. Cell lysates are cleared by centrifugation and stored at −80° C.

Western Blot analysis

In order to detect translation of transfected RNA, proteins from eithercell culture supernatants or cell lysates are separated by a 12%SDS-PAGE and blotted onto a nitrocellulose membrane. The humanisedantibody Herceptin (Roche) can be used as a control. After blotting iscompleted, membranes are consecutively incubated with a biotinylatedgoat anti-human IgG antibody (Dianova), streptavidin coupled tohorseradish peroxidase (BD), and a chemiluminescent substrate(SuperSignal West Pico, Pierce). Staining is detected with a FujiLAS-1000 chemiluminescence camera.

FACS analysis

Functional antibody formation can be demonstrated by FACS staining ofantigen-expressing target cells. In order to examine the production offunctional antibodies, cell culture supernatants of RNA-transfectedcells are collected after 48 to 96 hours. Approximately 200.000 targetBT-474 cells expressing HER2 are incubated with either controlantibodies (Herceptin, Roche) or cell culture supernatants. Fordetection of bound antibodies, cells are stained with biotinylated goatanti-human IgG (Dianova) and PE-labelled streptavidin (Invitrogen).Cells are analysed on a FACSCanto II (BD).

The invention claimed is:
 1. A method of expressing a therapeuticprotein in a mammalian subject comprising administering to the subjectan effective amount of mRNA comprising: a) a polypeptide coding region,encoding said therapeutic protein; b) at least one histone stem-loop,and c) a poly(A) sequence, wherein said mRNA does not include a histonedownstream element (HDE) and the mRNA increases the expression of thetherapeutic protein in said subject.
 2. The method of claim 1, whereinthe mRNA does not comprise a sequence encoding a reporter protein, amarker or selection protein.
 3. The method of claim 1, wherein thepoly(A) sequence comprises a sequence of about 25 to about 400 adenosinenucleotides.
 4. The method of claim 3, wherein the RNA comprises a 5′cap structure and a poly(A) sequence of about 25 to about 400 adenosinenucleotides.
 5. The method of claim 1, wherein the G/C content of thepolypeptide coding region is increased compared with the G/C content ofthe coding region of the wild-type nucleic acid encoding the therapeuticprotein.
 6. The method of claim 1, wherein the therapeutic protein isselected from the group consisting of an Acid sphingomyelinase,Adipotide, Agalsidase-beta, Alglucosidase, alpha-galactosidase A,alpha-glucosidase, alpha-L-iduronidase, alpha-N-acetylglucosaminidase,Amphiregulin, Angiopoietin, Betacellulin, Beta-glucuronidase, Bonemorphogenetic protein (BMP), CLN6 protein, Epidermal growth factor(EGF), Epigen, Epiregulin, Fibroblast Growth Factor (FGF), Galsulphase,Ghrelin, Glucocerebrosidase, GM-CSF, Heparin-binding EGF-like growthfactor (HB-EGF), Hepatocyte growth factor HGF, Hepcidin, Human albumin,increased loss of albumin, Idursulphase, Integrin aVβ3, Integrin aVβ5,Integrin a5β1, Iuduronate sulfatase, Laronidase,N-acetylgalactosamine-4-sulfatase (rhASB),N-acetylglucosamine-6-sulfatase, Nerve growth factor (NGF),Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3),Neurotrophin 4/5 (NT-4/5), Neuregulin, Neuropilin, Obestatin, PlateletDerived Growth factor (PDGF), TGF beta receptor, Thrombopoietin (THPO),Megakaryocyte growth and development factor (MGDF), Transforming Growthfactor (TGF), VEGF, Nesiritide, Trypsin, adrenocorticotrophic hormone(ACTH), Atrial-natriuretic peptide (ANP), Cholecystokinin, Gastrin,Leptin, Oxytocin, Somatostatin, Vasopressin, Calcitonin, Exenatide,Growth hormone (GH), somatotropin, Insulin, Insulin-like growth factor 1(IGF-1), Mecasermin rinfabate, IGF-1 analog, Mecasermin, Pegvisomant,Pramlintide, Teriparatide, Becaplermin, Dibotermin-alpha, gonadotropinreleasing hormone (GnRH), Octreotide, and keratinocyte growth factor(KGF).
 7. The method of claim 1, wherein the therapeutic protein isselected from the group consisting of a tissue plasminogen activator(tPA), Anistreplase, Antithrombin III (AT-III), Bivalirudin,Darbepoetin-alpha, Drotrecogin-alpha, Erythropoietin (EPO), Factor IX,Factor VIIa, Factor VIII, Lepirudin, Protein C concentrate, Reteplase,Streptokinase, Tenecteplase, Urokinase, Angiostatin, Anti-CD22immunotoxin, Denileukin diftitox, Immunocyanin, MPS(Metallopanstimulin), Aflibercept, Endostatin, Collagenase, Humandeoxy-ribonuclease I, dornase, Hyaluronidase, Papain, L-Asparaginase,Peg-asparaginase, Rasburicase, Human chorionic gonadotropin (HCG), Humanfollicle-stimulating hormone (FSH), Lutropin-alpha, Prolactin,alpha-1-Proteinase inhibitor, Lactase, Pancreatic enzyme, lipase,amylase, protease, Adenosine deaminase, Abatacept, Alefacept, Anakinra,Etanercept, Interleukin-1 (IL-1) receptor antagonist, Anakinra,Thymulin, TNF-alpha antagonist, Enfuvirtide, and Thymosin al.
 8. Themethod of claim 1, wherein the therapeutic protein is selected from thegroup consisting of 3F8, 8H9, Abagovomab, Adecatumumab, Afutuzumab,Alacizumab pegol, Alemtuzumab, Amatuximab, AME-133v, AMG 102, Anatumomabmafenatox, Apolizumab, Bavituximab, Bectumomab, Belimumab, Bevacizumab,Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab,Cantuzumab mertansine, Cantuzumab ravtansine, Capromab pendetide,Carlumab, Catumaxomab, Cetuximab, Citatuzumab bogatox, Cixutumumab,Clivatuzumab tetraxetan, CNTO 328, CNTO 95, Conatumumab, Dacetuzumab,Dalotuzumab, Denosumab, Detumomab, Drozitumab, Ecromeximab, Edrecolomab,Elotuzumab, Elsilimomab, Enavatuzumab, Ensituximab, Epratuzumab,Ertumaxomab, Ertumaxomab, Etaracizumab, Farletuzumab, FBTA05,Ficlatuzumab, Figitumumab, Flanvotumab, Galiximab, Galiximab, Ganitumab,GC1008, Gemtuzumab, Gemtuzumab ozogamicin, Girentuximab, Glembatumumabvedotin, GS6624, HuC242-DM4, HuHMFG1, HuN901-DM1, Ibritumomab,Icrucumab, ID09C3, Indatuximab ravtansine, Inotuzumab ozogamicin,Intetumumab, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab,Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab,Mapatumumab, Matuzumab, MDX-060, MEDI522, Mitumomab, Mogamulizumab,MORab-003, MORab-009, Moxetumomab pasudotox, MT103, Nacolomab tafenatox,Naptumomab estafenatox, Narnatumab, Necitumumab, Nimotuzumab,Nimotuzumab, Olaratumab, Onartuzumab, Oportuzumab monatox, Oregovomab,Oregovomab, PAM4, Panitumumab, Patritumab, Pemtumomab, Pertuzumab,Pritumumab, Racotumomab, Radretumab, Ramucirumab, Rilotumumab,Rituximab, Robatumumab, Samalizumab, SGN-30, SGN-40, Sibrotuzumab,Siltuximab, Tabalumab, Tacatuzumab tetraxetan, Taplitumomab paptox,Tenatumomab, Teprotumumab, TGN1412, Ticilimumab (=tremelimumab),Tigatuzumab, TNX-650, Tositumomab, Trastuzumab, TRB S07, Tremelimumab,TRU-016, TRU-016, Tucotuzumab celmoleukin, Ublituximab, Urelumab,Veltuzumab, Veltuzumab (IMMU-106), Volociximab, Votumumab, WX-G250,Zalutumumab, Zanolimumab, Efalizumab, Epratuzumab, Etrolizumab,Fontolizumab, Ixekizumab, Mepolizumab, Milatuzumab, Priliximab,Rituximab, Rontalizumab, Ruplizumab, Sarilumab, Vedolizumab,Visilizumab, Reslizumab, Adalimumab, Aselizumab, Atinumab, Atlizumab,Bertilimumab, Besilesomab, BMS-945429, Briakinumab, Brodalumab,Canakinumab, Canakinumab, Certolizumab pegol, Erlizumab, Fezakinumab,Golimumab, Gomiliximab, Infliximab, Mavrilimumab, Natalizumab,Ocrelizumab, Odulimomab, Ofatumumab, Ozoralizumab, Pexelizumab,Rovelizumab, SBI-087, SBI-087, Secukinumab, Sirukumab, Talizumab,Tocilizumab, Toralizumab, TRU-015, TRU-016, Ustekinumab, Ustekinumab,Vepalimomab, Zolimomab aritox, Sifalimumab, Lumiliximab, Rho(D) ImmuneGlobulin, Afelimomab, CR6261, Edobacomab, Efungumab, Exbivirumab,Felvizumab, Foravirumab, Ibalizumab, Libivirumab, Motavizumab,Nebacumab, Tuvirumab, Urtoxazumab, Bavituximab, Pagibaximab,Palivizumab, Panobacumab, PRO 140, Rafivirumab, Raxibacumab,Regavirumab, Sevirumab, Suvizumab, Tefibazumab, Abciximab, Atorolimumab,Eculizumab, Mepolizumab, Milatuzumab; Antithymocyte globulin,Basiliximab, Cedelizumab, Daclizumab, Gavilimomab, Inolimomab,Muromonab-CD3, Muromonab-CD3, Odulimomab, Siplizumab, Gevokizumab,Otelixizumab, Teplizumab, Bapineuzumab, Crenezumab, Gantenerumab,Ponezumab, R1450, Solanezumab Benralizumab, Enokizumab, Keliximab,Lebrikizumab, Omalizumab, Oxelumab, Pascolizumab, Tralokinumab,Blosozumab, CaroRx, Fresolimumab, Fulranumab, Romosozumab, Stamulumab,Tanezumab, and Ranibizumab.
 9. The method of claim 1, wherein at leastone guanosine, uridine, adenosine, or cytidine position of the nucleicacid molecule is substituted with an analogue of these nucleotidesselected from 2-amino-6-chloropurineriboside-5′-triphosphate,2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5 ′-triphosphate,5-bromouridine-5 ′-triphosphate, 5 -iodocytidine-5 ′-triphosphate, 5odouridine-5 ′-triphosphate, 5 -methylcytidine-5 ′-triphosphate,5-methyluridine-5′-triphosphate, 6-azacytidine-5′-triphosphate,6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, andxanthosine-5′-triphosphate.
 10. The method of claim 1, wherein the mRNAcomprises a sequence of at least 10 consecutive cytidines.
 11. Themethod of claim 1, wherein the mRNA further comprises a stabilizingsequence from the alpha globin 3′ UTR, positioned 3′ relative to thepolypeptide coding region of the mRNA.
 12. The method of claim 1,wherein the subject has a disease selected from the group consisting ofan infectious disease, cancer, disease of the blood and blood-formingorgans, endocrine or metabolic disease, disease of the nervous system,disease of the circulatory system, disease of the respiratory system,disease of the digestive system, disease of the skin and subcutaneoustissue, disease of the musculoskeletal system and connective tissue, anddisease of the genitourinary system.
 13. The method of claim 1, whereinthe subject has an erythrocyte deficiency and the therapeutic protein iserythropoietin.
 14. The method of claim 1, wherein the encodedtherapeutic protein is for treatment of metabolic or endocrinedisorders.
 15. The method of claim 1, wherein the encoded therapeuticprotein is for treatment of blood disorders, diseases of the circulatorysystem, diseases of the respiratory system, cancer or tumour diseases,infectious diseases or immunedeficiencies.
 16. The method of claim 1,wherein the encoded therapeutic protein is for hormone replacementtherapy.
 17. The method of claim 1, wherein the encoded therapeuticprotein is for in reprogramming of somatic cells.
 18. The method ofclaim 1, wherein the encoded therapeutic protein is an adjuvant orimmunostimulating protein.
 19. The method of claim 1, wherein theencoded therapeutic protein is an antibody.
 20. The method of claim 19,wherein the therapeutic protein comprises Ipilimumab.